Arthroscopic Irrigation Solution and Method for Peripheral Vasoconstriction and Inhibition of Pain and Inflammation

ABSTRACT

A method and solution for perioperatively inhibiting a variety of pain and inflammation processes during arthroscopic procedures. The solution preferably includes a vasoconstrictor that exhibits alpha-adrenergic activity and one or more additional pain and inflammation inhibitory agents at dilute concentration in a physiologic carrier, such as saline or lactated Ringer&#39;s solution. The solution is applied by continuous irrigation of a wound during a surgical procedure for peripheral vasoconstriction and inhibition of pain and/or inflammation while avoiding undesirable side effects associated with systemic application of larger doses of the agents.

This application is a continuation of U.S. application Ser. No.13/047,386, filed Mar. 14, 2011, which is a continuation of U.S.application Ser. No. 10/138,192 filed May 1, 2002, now U.S. Pat. No.7,973,068, which is a continuation-in-part of U.S. application Ser. No.09/839,633 filed Apr. 20, 2001, which is a continuation-in-part ofInternational Application PCT/US99/24672 filed Oct. 20, 1999 designatingthe United States and that claims the benefit of the filing date of U.S.Provisional Application Ser. No. 60/105,029 filed Oct. 20, 1998, thebenefit of the filing dates of which are hereby claimed under 35 U.S.C.§119 and §120.

I. FIELD OF THE INVENTION

The present invention relates to arthroscopic surgical irrigationsolutions and methods, and particularly for anti-inflammatory, anti-painand vasoconstrictive arthroscopic surgical irrigation solutions.

II. BACKGROUND OF THE INVENTION

Arthroscopy is a surgical procedure in which a camera, attached to aremote light source and video monitor, is inserted into an anatomicjoint (e.g., knee, shoulder, etc.) through a small portal incision inthe overlying skin and joint capsule. Through similar portal incisions,surgical instruments may be placed in the joint, their use guided byarthroscopic visualization. As arthroscopists' skills have improved, anincreasing number of operative procedures, once performed by “open”surgical technique, now can be accomplished arthroscopically. Suchprocedures include, for example, partial meniscectomies and ligamentreconstructions in the knee, shoulder acromioplasties and rotator cuffdebridements and elbow synovectomies. As a result of widening surgicalindications and the development of small diameter arthroscopes, wristand ankle arthroscopies also have become routine.

Throughout each arthroscopy, physiologic irrigation fluid (e.g., normalsaline or lactated Ringer's) is flushed continuously through the joint,distending the joint capsule and removing operative debris, therebyproviding clearer intra-articular visualization. U.S. Pat. No. 4,504,493to Marshall discloses an isomolar solution of glycerol in water for anon-conductive and optically clear irrigation solution for arthroscopy.

Alleviating pain and suffering in postoperative patients is an area ofspecial focus in clinical medicine, especially with the growing numberof out-patient operations performed each year. The most widely usedagents, cyclooxygenase inhibitors (e.g., ibuprofen) and opioids (e.g.,morphine, fentanyl), have significant side effects includinggastrointestinal irritation/bleeding and respiratory depression. Thehigh incidence of nausea and vomiting related to opioids is especiallyproblematic in the postoperative period. Therapeutic agents aimed attreating postoperative pain while avoiding detrimental side effects arenot easily developed because the molecular targets for these agents aredistributed widely throughout the body and mediate diverse physiologicalactions. Despite the significant clinical need to inhibit pain andinflammation, methods for the delivery of inhibitors of pain andinflammation at effective dosages while minimizing adverse systemic sideeffects have not been developed. As an example, conventional (i.e.,intravenous, oral, subcutaneous or intramuscular) methods ofadministration of opiates in therapeutic doses frequently is associatedwith significant adverse side effects, including severe respiratorydepression, changes in mood, mental clouding, profound nausea andvomiting.

Prior studies have demonstrated the ability of endogenous agents, suchas serotonin (5-hydroxytryptamine, sometimes referred to herein as“5-HT”), bradykinin and histamine, to produce pain and inflammation.Sicuteri, F., et al., Serotonin-Bradykinin Potentiation in the PainReceptors in Man, Life Sci. 4, pp. 309-316 (1965); Rosenthal, S. R.,Histamine as the Chemical Mediator for Cutaneous Pain, J. Invest.Dermat. 69, pp. 98-105 (1977); Richardson, B. P., et al., Identificationof Serotonin M-Receptor Subtypes and their Specific Blockade by a NewClass of Drugs, Nature 316, pp. 126-131 (1985); Whalley, E. T., et al.,The Effect of Kinin Agonists and Antagonists, Naunyn-Schmiedeb Arch.Pharmacol. 36, pp. 652-57 (1987); Lang, E., et al., Chemo-Sensitivity ofFine Afferents from Rat Skin In Vitro, J. Neurophysiol. 63, pp. 887-901(1990).

For example, 5-HT applied to a human blister base (denuded skin) hasbeen demonstrated to cause pain that can be inhibited by 5-HT₃ receptorantagonists. Richardson et al., (1985). Similarly, peripherally appliedbradykinin produces pain that can be blocked by bradykinin receptorantagonists. Sicuteri et al., 1965; Whalley et al., 1987; Dray, A., etal., Bradykinin and Inflammatory Pain, Trends Neurosci. 16, pp. 99-104(1993). Peripherally-applied histamine produces vasodilation, itchingand pain that can be inhibited by histamine receptor antagonists.Rosenthal, 1977; Douglas, W. W., “Histamine and 5-Hydroxytryptamine(Serotonin) and their Antagonists”, in Goodman, L. S., et al., ed., ThePharmacological Basis of Therapeutics, MacMillan Publishing Company, NewYork, pp. 605-638 (1985); Rumore, M. M., et al., Analgesic Effects ofAntihistaminics, Life Sci 36, pp. 403-416 (1985). Combinations of thesethree agonists (5-HT, bradykinin and histamine) applied together havebeen demonstrated to display a synergistic pain-causing effect,producing a long-lasting and intense pain signal. Sicuteri et al., 1965;Richardson et al., 1985; Kessler, W., et al., Excitation of CutaneousAfferent Nerve Endings In Vitro by a Combination of InflammatoryMediators and Conditioning Effect of Substance P, Exp. Brain Res. 91,pp. 467-476 (1992).

In the body, 5-HT is located in platelets and in central neurons,histamine is found in mast cells, and bradykinin is produced from alarger precursor molecule during tissue trauma, pH changes andtemperature changes. Because 5-HT can be released in large amounts fromplatelets at sites of tissue injury, producing plasma levels 20-foldgreater than resting levels (Ashton, J. H., et al., Serotonin as aMediator of Cyclic Flow Variations in Stenosed Canine Coronary Arteries,Circulation 73, pp. 572-578 (1986)), it is possible that endogenous 5-HTplays a role in producing postoperative pain, hyperalgesia andinflammation. In fact, activated platelets have been shown to exciteperipheral nociceptors in vitro. Ringkamp, M., et al., Activated HumanPlatelets in Plasma Excite Nociceptors in Rat Skin, In Vitro, Neurosci.Lett. 170, pp. 103-106 (1994). Similarly, histamine and bradykinin alsoare released into tissues during trauma. Kimura, E., et al., Changes inBradykinin Level in Coronary Sinus Blood After the ExperimentalOcclusion of a Coronary Artery, Am Heart J. 85, pp. 635-647 (1973);Douglas, 1985; Dray et al. (1993).

In addition, prostaglandins also are known to cause pain andinflammation. Cyclooxygenase inhibitors, e.g., ibuprofen, are commonlyused in non-surgical and post-operative settings to block the productionof prostaglandins, thereby reducing prostaglandin-mediated pain andinflammation. Flower, R. J., et al., Analgesic-Antipyretics andAnti-Inflammatory Agents; Drugs Employed in the Treatment of Gout, inGoodman, L. S., et al., ed., The Pharmacological Basis of Therapeutics,MacMillan Publishing Company, New York, pp. 674-715 (1985).Cyclooxygenase inhibitors are associated with some adverse systemic sideeffects when applied conventionally. For example, indomethacin orketorolac have well recognized gastrointestinal and renal adverse sideeffects.

As discussed, 5-HT, histamine, bradykinin and prostaglandins cause painand inflammation. The various receptors through which these agentsmediate their effects on peripheral tissues have been known and/ordebated for the past two decades. Most studies have been performed inrats or other animal models. However, there are differences inpharmacology and receptor sequences between human and animal species.There have been no studies conclusively demonstrating the importance of5-HT, bradykinin or histamine in producing postoperative pain in humans.

Furthermore, antagonists of these mediators currently are not used forpostoperative pain treatment. A class of drugs, termed 5-HT andnorepinephrine uptake antagonists, which includes amitriptyline, hasbeen used orally with moderate success for chronic pain conditions.However, the mechanisms of chronic versus acute pain states are thoughtto be considerably different. In fact, two studies in the acute painsetting using amitriptyline perioperatively have shown no pain-relievingeffect of amitriptyline. Levine, J. D., et al., Desipramine EnhancesOpiate Postoperative Analgesia, Pain 27, pp. 45-49 (1986); Kerrick, J.M., et al., Low-Dose Amitriptyline as an Adjunct to Opioids forPostoperative Orthopedic Pain: a Placebo-Controlled Trial Period, Pain52, pp. 325-30 (1993). In both studies the drug was given orally. Thesecond study noted that oral amitriptyline actually produced a loweroverall sense of well-being in postoperative patients, which may be dueto the drug's affinity for multiple amine receptors in the brain.

Amitriptyline, in addition to blocking the uptake of 5-HT andnorepinephrine, is a potent 5-HT receptor antagonist. Therefore, thelack of efficacy in reducing postoperative pain in thepreviously-mentioned studies would appear to conflict with the proposalof a role for endogenous 5-HT in acute pain. There are a number ofreasons for the lack of acute pain relief found with amitriptyline inthese two studies. (1) The first study (Levine et al., 1986) usedamitriptyline preoperatively for one week up until the night prior tosurgery whereas the second study (Kerrick et al., 1993) only usedamitriptyline postoperatively. Therefore, no amitriptyline was presentin the surgical site tissues during the actual tissue injury phase, thetime at which 5-HT is purported to be released. (2) Amitriptyline isknown to be extensively metabolized by the liver. With oraladministration, the concentration of amitriptyline in the operative sitetissues may not have been sufficiently high for a long enough timeperiod to inhibit the activity of postoperatively released 5-HT in thesecond study. (3) Since multiple inflammatory mediators exist, andstudies have demonstrated synergism between the inflammatory mediators,blocking only one agent (5-HT) may not sufficiently inhibit theinflammatory response to tissue injury.

There have been a few studies demonstrating the ability of extremelyhigh concentrations (1%-3% solutions—i.e., 10-30 mg per milliliter) ofhistamine₁ (H₁) receptor antagonists to act as local anesthetics forsurgical procedures. This anesthetic effect is not believed to bemediated via H₁ receptors but, rather, due to a non-specific interactionwith neuronal membrane sodium channels (similar to the action oflidocaine). Given the side effects (e.g., sedation) associated withthese high “anesthetic” concentrations of histamine receptorantagonists, local administration of histamine receptor antagonistscurrently is not used in the perioperative setting.

III. SUMMARY OF THE INVENTION

The present invention provides a solution comprising an alpha adrenergicreceptor agonist selected for peripheral vasoconstrictive activity andone or more additional agents in low concentrations directed atinhibiting locally the mediators of pain and/or inflammation in aphysiologic electrolyte carrier fluid. The invention also provides amethod for perioperative delivery of the irrigation solution containingthese agents directly to a surgical site, where it works locally at thereceptor and enzyme levels to preemptively limit pain and inflammationat the site. Due to the local perioperative delivery method of thepresent invention, a desired therapeutic effect can be achieved withlower doses of agents than are necessary when employing other methods ofdelivery (i.e., intravenous, intramuscular, subcutaneous and oral).

In a preferred embodiment, the alpha adrenergic receptor agonist (alsoreferred to herein as “alpha agonist” or “α agonist”) selected for useas the vasoconstrictor is an agonist that is primarily selective fordirect agonist activity at alpha receptors, and that has relativelyminor interaction with beta adrenergic receptors (also referred toherein as “beta receptors” or “β receptors”). Agonists that areprimarily selective for direct agonist activity at alpha receptors andthat have relatively minor interaction with beta receptors (“α selectiveagonists”) include agonists that (a) act primarily at α₁-receptors(“α₁-receptor selective agonists”), (b) act substantially atα₂-receptors (“α₂-receptor selective agonists”), or (c) act primarily atboth α₁-receptors and α₂-receptors (“mixed α₁ and α₂ agonists”).

More preferably, the alpha agonist selected for use in the presentinvention is a mixed α₁ and α₂ agonist, as long as such mixed agonistprovides vasoconstriction at the target tissue. Most preferably, thealpha agonist selected for use in the present invention has agonistactivity at both α_(2A)-receptors and α_(1A)-receptors with high potencyat both α_(1A) and α_(2A)-receptors, as long as such agonist providesvasoconstriction at the target tissue.

In one embodiment, the additional anti-pain/anti-inflammation agent(s)in the solution may include one or more agents selected from thefollowing classes of receptor antagonists and agonists and enzymeactivators and inhibitors, each class acting through a differingmolecular mechanism of action for pain and/or inflammation inhibition:(1) serotonin receptor antagonists; (2) serotonin receptor agonists; (3)histamine receptor antagonists; (4) bradykinin receptor antagonists; (5)kallikrein inhibitors; (6) tachykinin receptor antagonists, includingneurokinin₁ and neurokinin₂ receptor subtype antagonists; (7) calcitoningene-related peptide (CGRP) receptor antagonists; (8) interleukinreceptor antagonists; (9) inhibitors of enzymes active in the syntheticpathway for arachidonic acid metabolites, including (a) phospholipaseinhibitors, including PLA₂ isoform inhibitors and PLC_(γ) isoforminhibitors, (b) cyclooxygenase inhibitors, including non-selectivecyclooxygenase inhibitors and cyclooxygenase-2 (COX-2) selectiveinhibitors, and (c) lipoxygenase inhibitors; (10) prostanoid receptorantagonists including eicosanoid EP-1 and EP-4 receptor subtypeantagonists and thromboxane receptor subtype antagonists; (11)leukotriene receptor antagonists including leukotriene B₄ receptorsubtype antagonists and leukotriene D₄ receptor subtype antagonists;(12) opioid receptor agonists, including μ-opioid, δ-opioid, andκ-opioid receptor subtype agonists; (13) purinoceptor agonists andantagonists including P_(2X) receptor antagonists and P_(2Y) receptoragonists; (14) adenosine triphosphate (ATP)-sensitive potassium channelopeners; (15) mitogen-activated protein kinase (MAPK) inhibitors; (16)neuronal nicotinic acetylcholine receptor agonists; and (17) solublereceptors. Each of the above agents functions either as ananti-inflammatory agent and/or as an anti-nociceptive, i.e., anti-painor analgesic, agent. The selection of agents from these classes ofcompounds is tailored for the particular application.

A preferred solution for use in the present invention includes (a) acyclooxygenase inhibitor (most preferably a nonselective cyclooxygenaseinhibitor that also acts to inhibit lipoxygenase), (b) a serotonin₂antagonist and/or a histamine₁ antagonist (most preferably an agent thatexhibits both of these functions) and (c) an alpha adrenergic receptoragonist as a peripheral vasoconstrictor (more preferably an alphaagonist that is highly selective for alpha receptors without substantial(relatively little or no) interaction with beta receptors, and mostpreferably that is a mixed alpha-1 and alpha-2 agonist).

The present invention also provides a method for manufacturing amedicament compounded as a dilute irrigation solution for use incontinuously irrigating an operative site or wound during an operativeprocedure. The method entails dissolving in a physiologic electrolytecarrier fluid an α-receptor agonist selected for local vasoconstrictiveactivity, and one or more additional anti-pain/anti-inflammatory agents.The α-receptor agonist is included at a concentration of preferably nomore than 300,000 nanomolar, and more preferably no more than 75,000nanomolar, depending on the particular agent selected. Each additionalanti-inflammatory and/or analgesic agent is included at a concentrationof preferably no more than 100,000 nanomolar, and more preferably nomore than 10,000 nanomolar, except for cyclooxygenase inhibitors, whichmay be required at larger concentrations of preferably no more than500,000 nanomolar, and more preferably no more than 200,000, dependingon the particular inhibitor selected.

The method of the present invention provides for the delivery of adilute combination of multiple receptor antagonists and agonists andenzyme inhibitors and activators directly to a wound or operative site,during therapeutic or diagnostic procedures for the inhibition of painand/or inflammation. Since the active ingredients in the solution arebeing locally applied directly to the operative tissues in a continuousfashion, the drugs may be used efficaciously at extremely low dosesrelative to those doses required for therapeutic effect when the samedrugs are delivered orally, intramuscularly, subcutaneously orintravenously. As used herein, the term “local” encompasses applicationof a drug in and around a wound or other operative site, and excludesoral, subcutaneous, intravenous and intramuscular administration. Theterm “continuous” as used herein encompasses uninterrupted application,repeated application at frequent intervals (e.g., repeated boluses atfrequent intervals intraprocedurally), and applications which areuninterrupted except for brief cessations such as to permit theintroduction of other drugs or agents or procedural equipment, such thata substantially constant predetermined concentration is maintainedlocally at the wound or operative site.

The advantages of low dose applications of agents are three-fold. Themost important is the absence of systemic side effects that often limitthe usefulness of these agents. Additionally, the agents selected forparticular applications in the solutions of the present invention arehighly specific with regard to the mediators on which they work. Thisspecificity is maintained by the low dosages utilized. Finally, the costof these active agents per operative procedure is low.

The advantages of local administration of the agents via luminalirrigation or other fluid application are the following: (1) localadministration guarantees a known concentration at the target site,regardless of interpatient variability in metabolism, blood flow, etc.;(2) because of the direct mode of delivery, a therapeutic concentrationis obtained instantaneously and, thus, improved dosage control isprovided; and (3) local administration of the active agents directly toa wound or operative site also substantially reduces degradation of theagents through extracellular processes, e.g., first- and second-passmetabolism, that would otherwise occur if the agents were given orally,intravenously, subcutaneously or intramuscularly. This is particularlytrue for those active agents that are peptides, which are metabolizedrapidly. Thus, local administration permits the use of compounds oragents which otherwise could not be employed therapeutically. Forexample, some agents in the following classes are peptidic: bradykininreceptor antagonists; tachykinin receptor antagonists; opioid receptoragonists; CGRP receptor antagonists; and interleukin receptorantagonists. Local, continuous delivery to the wound or operative siteminimizes drug degradation or metabolism while also providing for thecontinuous replacement of that portion of the agent that may bedegraded, to ensure that a local therapeutic concentration, sufficientto maintain receptor occupancy, is maintained throughout the duration ofthe operative procedure.

Local administration of the solution perioperatively throughout asurgical procedure in accordance with the present invention produces apreemptive analgesic and/or anti-inflammatory effect. As used herein,the term “perioperative” encompasses application intraprocedurally, pre-and intraprocedurally, intra- and postprocedurally, and pre-, intra- andpostprocedurally. To maximize the preemptive anti-inflammatory and/oranalgesic effects, the solutions of the present invention are mostpreferably applied pre-, intra- and postoperatively. By occupying thetarget receptors or inactivating or activating targeted enzymes prior tothe initiation of significant operative trauma locally, the agents ofthe present solution modulate specific pathways to preemptively inhibitthe targeted pathologic process. If inflammatory mediators and processesare preemptively inhibited in accordance with the present inventionbefore they can exert tissue damage, the benefit is more substantialthan if given after the damage has been initiated.

Inhibiting more than one inflammatory or nociceptive mediator byapplication of the multiple agent solution of the present inventiondramatically reduces the degree of inflammation and pain. In oneembodiment, the irrigation solutions of the present invention includecombinations of drugs, each solution acting on multiple receptors orenzymes. The drug agents are thus simultaneously effective against acombination of pathologic processes, including pain and inflammation.The action of these agents is considered to be synergistic, in that themultiple receptor antagonists and inhibitory agonists of the presentinvention provide a disproportionately increased efficacy in combinationrelative to the efficacy of the individual agents. The synergisticaction of several of the agents of the present invention are discussed,by way of example, below in the detailed descriptions of those agents.

The solutions of the present invention may be suitably applied to bothhumans and non-human mammals. The methods and solutions of the presentinvention are preferably delivered locally and perioperatively to awound during an arthroscopic procedure. As used hereafter, the term“wound”, unless otherwise specified, is intended to include surgicalwounds and operative/interventional sites.

In a further aspect of the invention, the solutions of the invention mayalso be locally and perioperatively delivered during open surgicalprocedures on joints of the extremities, including but not limited tototal knee, hip, ankle, toe, shoulder, elbow, wrist and finger jointreplacements, the placement of implants into joints of the extremities,and for other surgical procedures on an extremity. As used herein,“extremity” refers to anatomic structures of the leg, including the hip,or of the arm, including the shoulder.

Used perioperatively, the solution should result in a clinicallysignificant decrease in operative site pain and inflammation relative tocurrently-used irrigation fluids, thereby decreasing the patient'spostoperative analgesic (i.e., opiate) requirement and, whereappropriate, allowing earlier patient mobilization of the operativesite. No extra effort on the part of the surgeon and operating roompersonnel is required to use the present solution relative toconventional irrigation fluids.

As noted above, perioperative delivery of the solutions of the presentinvention during surgical procedures is preferred for a preemptive painand/or inflammation inhibitory effect. In a further aspect of theinvention, solutions of the invention including an alpha-selectiveadrenergic receptor agonist and one or more additional analgesic oranti-inflammatory agents in a physiologic irrigation carrier may also beused for direct irrigation of wounds before (preoperative) and/or during(intraoperative) and/or after (postoperative) an arthroscopic procedure,an open surgical procedure on an extremity joint, or othersurgical/interventional procedure on an extremity.

In a still further aspect of the invention, solutions of the inventionincluding an alpha-selective adrenergic receptor agonist and one or moreadditional analgesic or anti-inflammatory agents in a physiologiccarrier may be administered by intraarticular or intracapsular injectionof joints. Such injectable solutions may include a sustained releasevehicle for extended therapeutic effect.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in greater detail, by way ofexample, with reference to the accompanying drawings in which:

FIGS. 1-3 provide charts showing the effects of agents that are suitablefor use in a solution of the present invention on inflammatorysolution-induced plasma extravasation in a rat knee joint model, asdescribed in Example VI. FIGS. 1, 2 and 3 illustrate the effects ofsingle agents, two agent combinations, and three agent combinations,respectively, as now described in greater detail.

FIG. 1 shows the effect of individual drugs on inflammatory solution(IS)-induced plasma extravasation. Baseline was established by perfusionwith saline for 5 min followed by saline (control; n=16), amitriptyline(control; n=6), ketoprofen (control; n=6), or oxymetazoline (control;n=6), for 10 min. The inflammatory solution then was added to theperfusion fluid. Values are mean±_(SEM); n=number of knees.

FIG. 2 shows the effect of two-drug combinations and the three-drugcombination on inflammatory solution-induced plasma extravasation.Baseline was established by perfusion with saline for 5 min followed bysaline (control; n=16), amitriptyline+ketoprofen (Ami+Ket; n=6),amitriptyline+oxymetazoline (Ami+Oxy; n=6), ketoprofen+oxymetazoline(Ket+Oxy; n=6), or amitriptyline+ketoprofen+oxymetazoline (AKO; n=10)for 10 min. The inflammatory solution (IS) then was added to theperfusion fluid. Values are mean±_(SEM); n=number of knees. *P<0.0001vs. control. **P<0.05 vs. Ket+Oxy.

FIG. 3 shows the effect of the three-drug combination (preemptive andpost-inflammatory) on inflammatory solution-induced plasmaextravasation. In the control (n=16), baseline was established byperfusion with saline for 15 min followed by the inflammatory solution(IS). In the preemptive group (AKO pre; n=10), saline was perfused for 5min (baseline) and amitriptyline+ketoprofen+oxymetazoline for the next10 min; then the inflammatory solution was added to the perfusion fluid.In the post-inflammatory group (AKO post; n=6), baseline was establishedby perfusion with saline for 15 min; then the inflammatory solution wasstarted, followed by the addition ofamitriptyline+ketoprofen+oxymetazoline after 10 min. Values aremean±_(SEM); n =number of knees.

V. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The irrigation solutions of the present invention are dilute solutionsincluding at least one α-receptor agonist selected for peripheral(local) vasoconstrictor activity, and one or more additionalpain/inflammation inhibitory agents in a physiologic carrier. Thecarrier is a liquid, which as used herein is intended to encompassbiocompatible solvents, suspensions, polymerizable and non-polymerizablegels, pastes and salves. Preferably the carrier is an aqueous solutionthat may include physiologic electrolytes, such as normal saline orlactated Ringer's solution.

In a preferred embodiment, the alpha adrenergic receptor agonist (alsoreferred to herein as “alpha agonist” or “α agonist”) selected for useas the vasoconstrictor is an agonist that is primarily selective fordirect agonist activity at alpha receptors, and that has relativelyminor interaction with beta adrenergic receptors (also referred toherein as “beta receptors” or “β receptors”).

Agonists that are primarily selective for direct agonist activity atalpha receptors and that have relatively minor interaction with betareceptors (“α selective agonists”) include agonists that (a) actprimarily at α₁-receptors (“α₁-receptor selective agonists”), (b) actprimarily at α₂-receptors (“α₂-receptor selective agonists”), or (c) actsubstantially at both α₁-receptors and α₂-receptors (“mixed α₁ and α₂agonists”).

More preferably, the alpha agonist selected for use in the presentinvention is a mixed α₁ and α₂ agonist, as long as such mixed agonistprovides vasoconstriction at the target tissue. Most preferably, thealpha agonist selected for use in the present invention has agonistactivity at both α_(2A)-receptors and α_(1A)-receptors with high potencyat both α_(1A) and α_(2A)-receptors, as long as such agonist providesvasoconstriction at the target tissue.

When the α-receptor agonist selected for peripheral vasoconstrictoractivity for use in the present invention has at least partial agonistactivity at alpha-2 receptors, this vasoconstrictive agent may alsofunction as an analgesic agent in the solution, and may play a role ininhibiting the vasodilation component of inflammation. These functionsare dependent on agonist concentration and the local tissue receptorcharacteristics.

The one or more additional anti-inflammation/anti-pain agents areselected from the group consisting of: (1) serotonin receptorantagonists; (2) serotonin receptor agonists; (3) histamine receptorantagonists; (4) bradykinin receptor antagonists; (5) kallikreininhibitors; (6) tachykinin receptor antagonists, including neurokinin₁and neurokinin₂ receptor subtype antagonists; (7) calcitoningene-related peptide (CGRP) receptor antagonists; (8) interleukinreceptor antagonists; (9) inhibitors of enzymes active in the syntheticpathway for arachidonic acid metabolites, including (a) phospholipaseinhibitors, including PLA₂ isoform inhibitors and PLC_(γ) isoforminhibitors, (b) cyclooxygenase inhibitors, including non-selectivecyclooxygenase inhibitors and cyclooxygenase-2 (COX-2) selectiveinhibitors, and (c) lipoxygenase inhibitors; (10) prostanoid receptorantagonists including eicosanoid EP-1 and EP-4 receptor subtypeantagonists and thromboxane receptor subtype antagonists; (11)leukotriene receptor antagonists including leukotriene B₄ receptorsubtype antagonists and leukotriene D₄ receptor subtype antagonists;(12) opioid receptor agonists, including μ-opioid, δ-opioid, andκ-opioid receptor subtype agonists; (13) purinoceptor agonists andantagonists including P_(2X) receptor antagonists and P_(2Y) receptoragonists; (14) adenosine triphosphate (ATP)-sensitive potassium channelopeners; (15) mitogen-activated protein kinase (MAPK) inhibitors; (16)neuronal nicotinic acetylcholine receptor agonists; and (17) solublereceptors.

In each of the surgical solutions of the present invention, the agentsare included in low concentrations and are delivered locally in lowdoses relative to concentrations and doses required with conventionalmethods of drug administration to achieve the desired therapeuticeffect. It is impossible to obtain an equivalent therapeutic effect bydelivering similarly dosed agents via other (i.e., intravenous,subcutaneous, intramuscular or oral) routes of drug administration sincedrugs given systemically are subject to first- and second-passmetabolism. The concentration of each agent is determined in part basedon its dissociation constant, K_(d.) As used herein, the termdissociation constant is intended to encompass both the equilibriumdissociation constant for its respective agonist-receptor orantagonist-receptor interaction and the equilibrium inhibitory constantfor its respective activator-enzyme or inhibitor-enzyme interaction.Each agent is preferably included at a low concentration of 0.1 to10,000 times K_(d), except for cyclooxygenase inhibitors, which may berequired at larger concentrations depending on the particular inhibitorselected. Preferably, each agent is included at a concentration of 1.0to 1,000 times K_(d) and most preferably at approximately 100 timesK_(d). These concentrations are adjusted as needed to account fordilution in the absence of metabolic transformation at the localdelivery site. The exact agents selected for use in the solution, andthe concentration of the agents, varies in accordance with theparticular application, as described below.

In one embodiment, the surgical solutions constitute a novel therapeuticapproach by combining multiple pharmacologic agents acting at distinctreceptor and enzyme molecular targets. To date, pharmacologic strategieshave focused on the development of highly specific drugs that areselective for individual receptor subtypes and enzyme isoforms thatmediate responses to individual signaling neurotransmitters andhormones. This standard pharmacologic strategy, although well accepted,is not optimal since many other agents simultaneously may be responsiblefor initiating and maintaining inflammation and pain. Furthermore,despite inactivation of a single receptor subtype or enzyme, activationof other receptor subtypes or enzymes and the resultant signaltransmission often can trigger a cascade effect. This explains thesignificant difficulty in employing a single receptor-specific drug toblock a pathophysiologic process in which multiple transmitters play arole. Therefore, targeting only a specific individual receptor subtype,such as ET_(A), is likely to be ineffective.

In contrast to the standard approach to pharmacologic therapy, thetherapeutic approach of the present surgical solutions is based on therationale that a combination of drugs acting simultaneously on distinctmolecular targets is required to inhibit the full spectrum of eventsthat underlie the development of a pathophysiologic state. Furthermore,instead of targeting a specific receptor subtype alone, the surgicalsolutions are composed of drugs that target common molecular mechanismsoperating in different cellular physiologic processes involved in thedevelopment of pain and/or inflammation. In this way, the cascading ofadditional receptors and enzymes in the nociceptive and inflammatorypathways is minimized by the surgical solutions. In thesepathophysiologic pathways, the surgical solutions inhibit the cascadeeffect both “upstream” and “downstream”.

An example of “upstream” inhibition is the cyclooxygenase antagonists inthe setting of pain and inflammation. The cyclooxygenase enzymes (COX₁and COX₂) catalyze the conversion of arachidonic acid to prostaglandinH, which is an intermediate in the biosynthesis of inflammatory andnociceptive mediators including prostaglandins, leukotrienes, andthromboxanes. The cyclooxygenase inhibitors block “upstream” theformation of these inflammatory and nociceptive mediators. This strategyprecludes the need to block the interactions of the seven describedsubtypes of prostanoid receptors with their natural ligands. A similar“upstream” inhibitor included in the surgical solutions is aprotinin, akallikrein inhibitor. The enzyme kallikrein, a serine protease, cleavesthe high molecular weight kininogens in plasma to produce bradykinins,important mediators of pain and inflammation. By inhibition ofkallikrein, aprotinin effectively inhibits the synthesis of bradykinins,thereby providing an effective “upstream” inhibition of theseinflammatory mediators. The surgical solutions also make use of“downstream” inhibitors to control the pathophysiologic pathways.

The agents included in the solutions of the present invention may bedelivered in combination with other small molecule drugs, peptides,proteins, recombinant chimeric proteins, antibodies, or gene therapyvectors (viral and nonviral) to the spaces of the joint. The agentsexert actions on any cells associated with the fluid spaces of the jointand structures comprising the joint, and which are involved in thenormal function of the joint or are present due to a pathologicalcondition. These cells and structures include, but are not limited to:synovial cells including both Type A fibroblast and type B macrophagecells; the cartilaginous components of the joint such as chondrocytes;cells associated with bone, including periosteal cells, osteoblasts,osteoclasts; the immunological components such as inflammatory cellsincluding lymphocytes, mast cells, monocytes, eosinophils; and othercells like fibroblasts; and combinations of the above cell types.

A. Vasoconstrictors

The solutions of the present invention, and methods for delivering thesesolutions, utilize a vasoconstrictor delivered locally andperioperatively together with at least one, and preferably acombination, of additional agents that are inhibitors of inflammation orpain.

1. Rationale for inclusion of a Vasoconstrictor

One rationale for including a vasoconstrictor agent is to increase theeffectiveness of local drug delivery during arthroscopic surgery. Theaddition of a vasoconstrictor increases duration of action, decreaseslocal bleeding and potentially decreases systemic toxic reactions. Theability to combine a vasoconstrictor with other anti-pain andanti-inflammatory agents in the perioperative setting provides severaladvantages, including potentially decreased analgesia andanti-inflammatory drug usage, and decreased surgical blood loss. Controlof bleeding and improved visualization of the operative field is crucialto the outcome of endoscopic surgery. Certain operative procedures, suchas arthroscopy of the knee, often employ the use of a tourniquet tominimize bleeding. The inclusion of the vasoconstrictor in theirrigation fluid may reduce the need for a tourniquet in suchprocedures. Using a vasoconstrictor agent reduces vascular uptake anddelays clearance of the locally delivered drug combination, thusprolonging the anti-pain and anti-inflammatory effects. Furthermore, useof the vasoconstrictor has the potential to reduce peak blood plasmaconcentrations of the locally delivered combination of agents, thusenabling a higher maximum concentration of each drug to be delivered inthe irrigation fluid without a concomitant increase in blood levels. Inaddition, because vasodilation is a well accepted component ofinflammation, a vasoconstrictor can play a role in inhibiting thiscomponent of inflammation.

2. Adrenergic Receptor Agonists

Agonists that act on alpha adrenergic receptors exhibit vasoconstrictiveactivity. Certain types of adrenergic receptor agonists are suitable foruse as vasoconstrictors in the solutions and methods of the presentinvention, for local perioperative delivery together with one or moreanti-inflammatory or analgesic agents. Adrenergic receptor agonists thatare selective for alpha-1 receptors are well established as havingvasoconstrictive effects. Adrenergic receptor agonists that areselective for alpha-2 receptors are generally considered to havevasodilator effects. However, in some tissues exhibiting acute orchronic inflammation, such as in the knee, adrenergic receptor agoniststhat are active at alpha-2 receptors may have a vasoconstrictive effect.Adrenergic receptor agonists exhibiting at least partial agonistactivity at alpha-2 receptors may also have analgesic and/oranti-inflammatory effects. In near-normal knees (for example), alpha-1selective agonists may suitably be used as vasoconstrictors in thepresent invention. In knees (for example) exhibiting chronic or acuteinflammation, or subject to surgical trauma, an agonist with alpha-2activity or partial selectivity (e.g., mixed alpha-1 and alpha-2agonists) may be preferred for use in the present invention, and suchagonists will also provide analgesic and/or anti-inflammatory effect.

Before further describing the characteristics of preferred adrenergicagents, some background may be useful. All the individual nine receptorsthat comprise the adrenergic amine receptor family belong to theG-protein linked superfamily of receptors. The classification of theadrenergic family into three distinct subfamilies, namely α₁, α₂, and β,is based upon a wealth of binding, functional and second messengerstudies. Each adrenergic receptor subfamily is itself composed of threehomologous receptor subtypes that have been defined by cloning andpharmacological characterization of the recombinant receptors. Amongadrenergic receptors in different subfamilies (α₁ vs. α₂ vs. β), aminoacid identities in the membrane spanning domain range from 36-73%.However, between members of the same subfamily (α_(1A) vs. α_(1B)) theidentity between membrane domains is usually 70-80%. Together, thesedistinct receptor subtypes mediate the effects of two physiologicalagonists, epinephrine and norepinephrine.

Distinct adrenergic receptor types couple to unique sets of G-proteinsand are thereby capable of activating different signal transductioneffectors. The classification of α₁, α₂, and β subfamilies not onlydefines the receptors with regard to signal transduction mechanisms, butalso accounts for their ability to differentially recognize variousnatural and synthetic adrenergic amines. In this regard, a number ofselective ligands have been developed and utilized to characterize thepharmacological properties of each of these receptor types. Functionalresponses of α₁-receptors have been shown in certain systems tostimulate phosphatidylinositol turnover and promote the release ofintracellular calcium (via G_(q)), while stimulation of α₂-receptorsinhibits adenylate cyclase (via G_(i)). In contrast, functionalresponses of β-receptors are coupled to increases in adenylate cyclaseactivity and increases in intracellular calcium (via G_(s)).

It is now accepted that there are three different α₁ receptor subtypeswhich all exhibit a high affinity (subnanomolar) for the antagonistprazosin. The subdivision of α₁-adrenoceptors into three differentsubtypes, designated α_(1A), α_(1B), and α_(1D), has been primarilybased on extensive ligand binding studies of endogenous receptors andcloned receptors. Pharmacological characterization of the clonedreceptors led to revisions of the original classification such that theclone originally called the α_(1C) subtype corresponds to thepharmacologically defined α_(1A) receptor. Agonist occupation ofα_(1A-D)-receptor subtypes results in activation of phospholipase C,stimulation of PI breakdown, generation of the IP₃ as second messengerand an increase in intracellular calcium.

Three different α₂-receptor subtypes have been cloned, sequenced, andexpressed in mammalian cells, referred to as α_(2A) (α₂-C10), α_(2B)(α₂-C2), α_(2C) (α₂-C4). These subtypes not only differ in their aminoacid composition but also in their pharmacological profiles anddistributions. An additional α₂-receptor subtype, α_(2D) (gene rg20),was originally proposed based on radioligand binding studies of rodenttissues but is now considered to represent a species homolog to thehuman α_(2A)-receptor.

Functionally, the signal transduction pathways are similar for all threeα_(2A)-receptor subtypes; each is negatively coupled to adenylatecyclase via G_(i/o). In addition, the α_(2A) and α_(2B)-receptors havealso been reported to mediate activation of a G-protein coupledpotassium channel (receptor-operated) as well as inhibition of aG-protein associated calcium channel.

Pharmacologically, α₂-adrenergic receptors are defined as highlysensitive to the antagonists yohimbine (Ki=0.5-25 μM), atipamezole(Ki=0.5-2.5 μM), and idazoxan (Ki=21-35 μM) and with low sensitivity tothe α₁-receptor antagonist prazosin. Agonists selective for theα₂-adrenergic receptor class relative to the α₁-adrenergic receptorclass are UK14304, BHT920 and BHT933. Oxymetazoline binds with highaffinity and selectivity to the α_(2A)-receptor subtype, but in additionbinds with high affinity to α₁-adrenergic receptors and 5HT1 receptors.An additional complicating factor is that α₂-adrenergic receptor ligandsthat are imidazolines (clonidine, idazoxan) and others (oxymetazolineand UK14304) also bind with high affinity (nanomolar) tonon-adrenoceptor imidazoline binding sites. Furthermore, speciesvariation in the pharmacology of the α_(2A)-adrenoceptor exists. Todate, subtype-selective α₂-adrenergic receptor ligands show only minimalselectivity or are nonselective with respect to other specificreceptors, such that the therapeutic properties of subtype selectivedrugs are still under development.

3. Role of Alpha Receptors in the Regulation of Blood Flow

A study has been carried out to examine the effect of acute inflammationon the response of articular blood vessels in the knee joints of rabbitsto adrenergic receptor agonists. Gray E, et al, Ann. Rheum. Dis.,51(10):1129-33 (1992). Acute joint inflammation was found to alter theresponse of articular blood vessels in the knee joints of rabbits tovarious adrenoceptor agonists. The responses to noradrenaline,phenylephrine, clonidine, UK-14304, and isoprenaline were examined 24hours after intra-articular carrageenan injection and compared withthose of normal animals. Antagonists specific for alpha-1 and alpha-2receptors were used to identify the adrenergic receptors through whichthe responses were mediated, and to examine if carrageenan treatmentaltered the receptor response profile of these blood vessels. The studyreported that, in the carrageenan treated animals, there is a reductionin the alpha-1 response with an associated increase in the alpha-2response. A decrease in the number or affinity of alpha-1 adrenergicreceptors is indicated by a shift to the right of the noradrenaline andphenylephrine dose/response-curves, whereas an increase in alpha-2affinity or number is suggested by an associated leftward shift in thealpha-2 adrenoceptor agonist curves. This change in receptor responseprofile appears to arise as a direct result of carrageenan-induced jointinflammation. Clonidine, a mixed α₂ and α₁ agonist with greater α₂activity, nearly produced no response in normal animals, whereas in theinflammatory knee a distinct dose-dependent vasoconstriction wasobserved. Based on this study and similar studies, drugs possessingmixed α₂ and α₁ receptor selectivity profiles activate alpha-1adrenergic receptors that mediate the vasoconstrictor response withinsynovial blood vessels of the normal knee, as well as alpha-2 receptorsthat become important in the injured or inflamed knee. While clonidinehas weak alpha-1 activity, other mixed alpha-1 and alpha-2 agonists,such as oxymetazoline, will exhibit both significant alpha-1 and alpha-2effect. In a near normal knee, a preferred agent for use in irrigationsolutions will be an alpha-1 selective agent, or a mixed alpha-1 andalpha-2 agent with strong alpha-1 effect. However, a preferred agent toachieve vasoconstriction for use in arthroscopic irrigating solutions ofthe present invention, particularly for use in an injured knee with apre-existing acute or chronic inflammatory state, may be a mixed alpha-1and 2 receptor agonist having potent alpha-2 selective activity.

It has been shown that chronic inflammation may cause a reduction insympathetic nerve-mediated vasoconstriction in rat knees. McDougall (AmJ Physiol Regul Integr Comp Physiol, 281(3):R821-7 (2001)) studied therole of alpha-adrenergic receptors in regulating vasoactivity inchronically inflamed rat knee joints. To determine whether thisphenomenon is due to an alteration in smooth muscle adrenoceptorfunction, the study compared the alpha-adrenoceptor profile of bloodvessels supplying the anteromedial capsule of normal and chronicallyinflamed rat knee joints. The alpha-1 adrenoceptor agonists methoxamineand phenylephrine and the predominantly alpha-2 adrenoceptor agonistclonidine (0.1 ml bolus; dose range 10⁻¹²-10⁻⁷ mol) were applied toexposed normal rat knees, resulting in a dose-dependent fall in capsularperfusion. One week after intra-articular injection of Freund's completeadjuvant (FCA) to induce chronic joint inflammation, the vasoconstrictoreffects of methoxamine, phenylephrine, and clonidine were allsignificantly attenuated compared with normal controls. These changesmay be due to an increase in sympathetic nerve hyperactivity that leadsto a substantial increase in norepinephrine in the knee, resulting invasoconstriction. Because norepinepherine acts on both alpha-1 andalpha-2 receptors, the continuous activation may lead to chronicsympathetic hyperactivity and downregulation and/or desensitization ofall alpha-receptors binding sites in the joint.

In summary, knee blood vessels possess vasoconstrictor tone due to basalactivation of alpha-1 and alpha-2 receptors. Thus, both acute andchronic joint inflammation alter the alpha-1 and alpha-2 adrenergicreceptor response profile, and this change in alpha-adrenergicpharmacologic responsiveness provides a rationale for the therapeuticadvantage obtained using a mixed alpha agonist with significant alpha-2agonist efficacy for irrigation associated with arthroscopic proceduresfollowing joint trauma.

A variety of other studies confirm that vasoconstrictive responses inthe chronically inflamed knee are locally mediated by both vascularalpha-1 and -2 adrenergic receptors. A study of blood flow changes inresponse to selected alpha-1 and alpha-2 adrenoceptor agonists comparedinflamed and untreated knee joints using the laser Doppler flowmetery(LDF) technique. Badavi, M. et al, Exp. Physiol., 85(1):49-55 (2000).Yohimbine (an alpha-2 antagonist) was injected (0.5 mg kg-1, I.P.) 30min before phenylephrine application. Yohimbine blocked thevasoconstrictor effect of 10⁻¹⁰-10⁻⁷ molar clonidine (topicalapplication) by 44-67.7% inhibition. Prazosin (an alpha1 antagonist)blocked the vasoconstrictor effect of phenylephrine (10⁻¹⁰-10⁻⁷ molar,topical application) effectively (42 to 69.8% inhibition). This studyalso confirms the need and therapeutic advantage of using a mixedalpha-1 and alpha-2 agonist.

4. Adrenergic Drug Selectivity

Drug selectivity is extremely important in relation to pharmacologicaleffects mediated by drugs affecting the sympathetic nervous system andthe vasculature. Selectivity of a drug is typically a concentration- ordose-dependent property of a particular drug. At high concentration, aso-called selective drug may stimulate more than one type ofreceptor(s). The concentration at which this loss of selectivity occursis used as a measurement for this aspect of a drug's selectivitycharacteristics, and when comparing the selectivity of one drug to thatof another, often is expressed as a ratio of each drug's IC₅₀ or K_(d).For example, epinephrine stimulates the two main types of adrenergicreceptors, alpha and beta, and the subtypes in each family. In thisrespect, epinephrine is a non-selective alpha and beta agonist.

The selectivity of a drug used as a vasoconstrictor in an arthroscopicsurgical procedure not only affects its action in the joint, but alsoplays a role in the side-effects it produces elsewhere in the body onadrenergic receptors (e.g., the heart, lungs stomach, urinary bladder,ureter and uterus), to the extent that there is any systemic uptake ofthe drug. The chief vascular actions of epinephrine, a non-selective aand agonist, for example, are exerted on the smaller arterioles andprecapillary sphincters, although veins and arteries also respond to thedrug. Vascular beds vary in their response to the drug, depending on therate of infusion, the dose, and the ratio of alpha to beta adrenergicreceptor types and responses characteristic of the various vascularbeds. Total peripheral vascular resistance may drop due to beta-2receptor vasodilation action, which is opposed by vasoconstrictor actionmediated through alpha receptors. A vasoconstrictor drug possessingenhanced selectivity for alpha receptors relative to beta receptorswould minimize beta mediated receptor responses, including potentialcardiac toxicity, and thus is preferred for use in the presentinvention.

5. Pharmacology of Oxymetazoline

Oxymetazoline is an adrenergic receptor agonist that has highselectivity for alpha receptors relative to beta receptors, and is apreferred vasoconstrictive agent for use in the present invention.Oxymetazoline is relatively unique in its receptor selectivity profileand pharmacological efficacy and kinetic characteristics. Interestingly,it can be defined as a high affinity/low efficacy mixed alpha adrenergicagonist. In many tissues, it is a highly potent, partial agonist actingon alpha-2 receptors. Binding constants and activation constants foroxymetazoline are in the low nanomolar range for this receptor target.Among the alpha-2 receptor subtypes, oxymetazoline is specific in itsinteraction with the alpha-2A subtype. In addition, oxymetazoline alsodemonstrates alpha-1 receptor agonist activity, demonstratingspecificity for the alpha 1 subtypes. Unlike epinephrine, oxymetazolinedoes not interact with the beta-adrenergic receptor family. Within theadrenergic receptor family, oxymetazoline is a partial agonist atalpha-2A receptors and an agonist at alpha-1A receptors. Based uponbinding affinities determined by competition at cloned alpha-1 subtypemembrane preparations, oxymetazoline shows much higher affinity(selectivity) for the alpha-1A subtype compared to the alpha-1B and thealpha-1D subtypes.

The selectivity of alpha-2 receptor agonist mechanisms accounts for useof these agonists as vasoconstrictors. Oxymetazoline is preferable toepinephrine for use in the present invention because it is essentiallyfree of beta receptor mediated direct cardiac effects associated withepinephrine, and, through its vasoconstrictor activity, will have asignificantly longer duration of action in prolonging the effectivenessof local drug delivery. Thus, oxymetazoline exhibits long acting localeffects and is less cardiotoxic due to its receptor specificity profile.Additionally, because oxymetazoline is highly water soluble, it is lessable to cross the blood-brain barrier and is more likely to have effectson the periphery, which is desirable.

Among the many known alpha-2 agonists, oxymetazoline is relativelyunique in its receptor specificity profile. For example, both clonidineand UK14303 (brimonidine) are non-subtype selective agonists among themembers of the alpha-2 receptor family. Following injury andinflammation, the peripheral excitability of nociceptor nerve endings isattenuated by locally delivered alpha-2 agents due to a peripheralmechanism. Alpha-1 selective and beta selective agonists do not mimicthe anti-nociceptive property inherent in alpha-2 agonists.

In addition, there also is evidence for interaction of oxymetazoline atan imidazoline binding site or sites in various tissues based uponradioligand binding studies. The signal transduction pathway and theendogenous ligand for the putative one or more imidazoline receptorsremains speculative since the imidazoline receptors are not yet fullycharacterized. It appears that imidazolines, such as oxymetazoline, mayinteract with both types of receptors, namely the alpha adrenergicreceptors and the imidazoline receptor. Other drugs, such as clonidineand cirazoline share this dual receptor specificity, and such drugs aresuitable vasoconstrictors for use in the present invention. Clonidine isan alpha-2 receptor agonist with some alpha-1 agonist properties. Whilenot wishing to be limited by theory, this may provide an additionalbasis for the activity associated with oxymetazoline's vasoconstrictiveaction.

Another advantage of oxymetazoline for use in the procedures of thepresent invention is its long duration of action after initial exposureto the tissue. This long duration of action does not depend upon thecontinued presence of the drug at therapeutic concentrations. Rather, itappears to be a result of the oxymetazoline-induced receptordesensitization. This property provides for continued action after thecompletion of the surgical procedure. For example, topical intranasalapplication of oxymetazoline results in constriction of dilatedarterioles and reduction in nasal blood flow. Reigle, et al.,Laryngoscope 102: 800-823, (1992). Following intranasal application ofoxymetazoline solutions, local vasoconstriction occurs within 5-10minutes and persists for 5-6 hours with a gradual decline over theensuing 6 hours.

For purposes of therapeutic selectivity in intraoperative irrigationfluids, the high affinity/low efficacy properties of oxymetazoline couldbe important since high affinity/low efficacy agonists theoreticallyhave a much greater potential for tissue selectivity. Kenakin studiedthe relative contribution of affinity and efficacy to the selectivity ofaction to oxymetazoline. Kenakin, Br. J. Pharmacol., 81(1):131-41(1984). Oxymetazoline demonstrated a pronounced tissue selectivity, whencompared to noradrenaline, by being a potent full agonist in ratanococcygeus muscle and a partial agonist in rat vas deferens. Schildanalysis with phentolamine, corynanthine, prazosin and yohimbineindicated no alpha-adrenoceptor heterogeneity within the ratanococcygeus muscle or between this tissue and rat vas deferens.Measurement of agonist K_(d) values and Schild analysis ofoxymetazoline's ability to inhibit the effects of noradrenaline (afteralkylation) confirmed the homogeneity of alpha-adrenoceptors withrespect to these two agonists. The above profiles of activity werepredicted based upon modeling that oxymetazoline had a higher affinitybut lower efficacy than noradrenaline. Experimentally, it was confirmedthat oxymetazoline had 5 times the affinity but 0.2 to 0.3 times theefficacy of noradrenaline, thus defining its partial agonist activity inthis tissue. It also behaves as a partial agonist in the rat aorta,producing only about 75% of the maximal response of phenylephrine.

6. Disadvantages of Epinephrine

While epinephrine is an adrenergic receptor agonist that exhibitsvasoconstrictive activity, it is not preferred for use in the presentinvention. The use of epinephrine has some limitations due to itsnon-specific alpha and beta adrenergic activities and associatedpotential dose-related cardiac and local toxic effects. In particular,in the operative setting, epinephrine injected into patients havinggeneral anesthesia with various inhalational agents, may cause cardiacdysrhythmias, the most important being ventricular fibrillation. Addingepinephrine also introduces the possibility of a drug interaction withtricyclic antidepressants and nonselective beta adrenergic blockers. Onepreferred anti-pain/anti-inflammatory agent for use in the presentinvention is amitriptyline. Amitriptyline is a member of the tricyclicgroup, and thus it is preferable not to use epinephrine in combinationwith amitriptyline.

Use of a vasoconstrictor in an aqueous irrigation solution (for example)requires the use of a drug with the requisite stability in aqueoussolutions. Epinephrine is unstable in alkaline solutions. When exposedto air or light, it turns pink from oxidation to adrenochrome and thenbrown from formation of polymers. In aqueous solution, epinephrinebreakdown depends on pH, light, temperature and oxidating agents, andthe breakdown increases with time. Oxidative instability is a potentialliability that is overcome by the use of a more stable vasoconstrictor,e.g., oxymetazoline. Also, acidic sodium metabisulphite hasconventionally been added to epinephrine solutions to preventepinephrine oxidation associated with its chemical instability. Forapplications involving a combination of agents, a potential requirementfor inclusion of metabisulphite in the multiple drug solution wouldprevent the use of many other desirable drugs due to undesirablechemical interactions with metabisulphite. Other vasoconstrictiveagents, such as oxymetazoline, do not require inclusion of sodiummetabisulphite, and there are no chemical-drug interactions that limitits use.

Based on the above factors, preferred alpha receptor agonists for use asvasoconstrictors in the present invention may include, depending on theapplication, alpha-1 selective agonists, alpha-2 selective agonists, andmixed alpha-1 and alpha-2 agonists. Suitable alpha-2 selective agonistsare discussed below relative to their co-function of providing analgesicand potentially anti-inflammatory effects.

Suitable alpha-1 selective agonists include phenylephrine, methoxamineand cirazoline, by way of example. Suitable concentrations (as deliveredlocally) for these agents when used as vasoconstrictors in the solutionsof the present invention are provided in Table 1. Suitable mixed alpha-1and alpha-2 agonists include, by way of example: oxymetazoline,p-aminoclonidine, naphazoline, tetrahydrozoline, anatazoline,tramazoline, monoxidine, apraclonidine (iopidine), guanfacine, guanabenzand xylazine. These and other mixed alpha-1 and alpha-2 agonists may bepreferred for perioperative delivery during arthroscopic surgery onacute or chronically inflamed joints. Suitable concentrations (asdelivered locally) for exemplary mixed alpha-1 and alpha-2 agonists whenused as vasoconstrictors in the solutions of the present invention arealso provided in Table 1.

TABLE 1 Exemplary Alpha-1 Selective and Mixed Alpha-1 and Alpha-2Adrenergic Receptor Agonist Vasoconstrictors Therapeutic TherapeuticMost Acceptable Efficient Preferred Preferred ConcentrationsConcentrations Concentrations Concentration Compounds (nM) (nM) (nM)(nM) Mixed α-1/α-2: p-aminoclonidine 0.002-200,000 0.01-50,0000.1-10,000 10-2,000 naphazoline 0.002-200,000 0.01-50,000 0.1-10,000100-10,000 oxymetazoline 0.001-100,000 0.01-25,000 0.05-15,000  5-10,000 xylazine 0.015-300,000 0.06-75,000 0.6-25,000  5-10,000 α-1selective: phenylephrine 0.001-200,000 0.02-50,000 0.1-25,000 200-20,000cirazoline 0.001-100,000 0.02-50,000 0.1-25,000 200-20,000 methoxamine0.001-200,000 0.02-50,000 0.1-25,000 500-20,000

B. Anti-inflammatory and Analgesic Agents

The following is a description of exemplary suitableanti-inflammation/anti-pain agents for combination with avasoconstrictor selected in accordance with the present invention.Preferably at least one anti-inflammation/anti-pain agent is selectedthat acts on a different receptor or molecular target than the selectedvasoconstrictive agent. More preferably multipleanti-inflammation/anti-pain agents are selected, each acting on adifferent receptor or molecular target than the otheranti-inflammation/anti-pain agent(s) and the selected vasoconstrictiveagent in the solution. The exact agents selected for a given solution inaccordance with the present invention will be determined by theapplication and the associated pain and inflammation actions. While notwishing to be limited by theory, the justification behind the selectionof the various classes of agents that is believed to render the agentsoperative is also set forth.

1. Alpha-2 Adrenergic Receptor Agonists

Alpha receptor agonists have been discussed above with respect to theirpotential function as vasoconstrictive agents when applied locally inthe setting of chronic or acute inflammation, an example of which is thelocal trauma associated with a surgical procedure. Alpha receptoragonists that are selective for alpha-2 receptors or having at leastpartial agonist activity at alpha-2 receptors also serve the dualfunction of being anti-pain/anti-inflammation agents. A therapeuticfield in which α₂-receptor agonists may be considered to have potentialuse is as an adjunct to anesthesia, for the control of pain and blockadeof neurogenic inflammation. Sympathetic nervous system stimulationreleases norepinephrine after tissue injury, and thus influencesnociceptor activity. α₂-receptor agonists, such as clonidine, caninhibit norepinephrine release at terminal nerve fiber endings and thusmay induce analgesia directly at peripheral sites (without actions onthe CNS). The ability of primary afferent neurons to releaseneurotransmitters from both their central and peripheral endings enablesthem to exert a dual, sensory and “efferent” or “local effector”function. The term, neurogenic inflammation, is used to describe theefferent function of the sensory nerves that includes the release ofsensory neuropeptides that contribute to the inflammatory process.Agents that induce the release of sensory neuropeptides from peripheralendings of sensory nerves, such as capsaicin, produce pain, inflammationand increased vascular permeability resulting in plasma extravasation.Drugs that block release of neuropeptides (substance P, CGRP) fromsensory endings are predicted to possess analgesic and anti-inflammatoryactivity. This mechanism of action has been established for other drugsthat exhibit analgesic and anti-inflammatory action in the periphery,such as sumatriptan and morphine, which act on 5HT1 and μ-opioidreceptors, respectively. Both of these drugs are agonists that activatereceptors that share a common mechanism of signal transduction with theα₂-receptors. UK14304 (brimonidine), like sumatriptan, has been shown toblock plasma extravasation within the dura mater through a prejunctionalaction on α₂-receptors.

Evidence supporting a peripheral analgesic effect of clonidine wasobtained in a study of the effect of intra-articular injection of thedrug at the end of an arthroscopic knee surgery. Gentili, M et al Pain64: 593-596 (1996). Clonidine is considered to exhibit nonopiateantinociceptive properties, which might allow its use as an alternativefor postoperative analgesia. In a study undertaken to evaluate theanalgesic effects of clonidine administered intravenously to patientsduring the postoperative period, clonidine was found to delay the onsetof pain and decrease the pain score. Thus, a number of studies havedemonstrated intra- and postoperative analgesia effects from drugsacting either at α₂-adrenergic receptors, indicating these receptors aregood therapeutic targets for new drugs to treat pain.

From the molecular and cellular mechanism of action defined forα₂-receptor agonists, such as UK14304, these compounds are expected toexhibit anti-nociceptive action on the peripheral terminals of primaryafferent nerves when applied intraoperatively in an irrigation solutiondirectly to a tissue or a joint. In particular, an α₂-receptor agonistis expected to be an effective drug delivered to a joint by anirrigation solution during an arthroscopic surgical procedure(periprocedurally). The α₂-receptor agonist may be delivered alone, orin combination with other small molecule drugs, peptides, proteins,recombinant chimeric proteins, antibodies, or gene therapy vectors(viral and nonviral) to the fluid spaces of the joint. The α₂-receptoragonist can exert its actions on any cells associated with the fluidspaces of the joint and structures comprising the joint and are involvedin the normal function of the joint or are present due to a pathologicalcondition. These cells and structures include, but are not limited to:synovial cells including both Type A fibroblast and type B macrophagecells; the immunological components such as inflammatory cells includinglymphocytes, mast cells, monocytes, eosinophils; and other cells likefibroblasts and vascular endothelial cells; and combinations of theabove.

α₂-receptors agonists are suitable for use in the current invention,delivered locally and perioperatively either as a single agent orpreferably in combination with other anti-pain and/or anti-inflammatorydrugs, to inhibit pain and inflammation. Representative α₂-receptorsagonists for the practice of the present invention include, for example:clonidine; dexmedetomidine; oxymetazoline;((R)-(−)-3′-(2-amino-1-hydroxyethyl)-4′-fluoro-methanesulfoanilide(NS-49); 2-[(5-methylbenz-1-ox-4-azin-6-yl)imino]imidazo line(AGN-193080); AGN 191103 and AGN 192172, as described in Munk, S. etal., J. Med. Chem. 39: 3533-3538 (1996);5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304,brimonidine);5,6,7,8-tetrahydro-6-(2-propenyl)-4H-thiazolo[4,5-d]azepin-2-amine(BHT920); 6-ethyl-5,6,7,8-tetrahydro-4H-oxaazolo[4,5-d]azepin-2-amine(BHT933), 5,6-dihydroxy-1,2,3,4-tetrahydro-1-naphyl-imidazoline(A-54741). Suitable concentrations for these agents when used in thepresent invention are set forth in Table 2.

TABLE 2 Exemplary Alpha-2 selective and Mixed Alpha-1 and Alpha-2Adrenergic Receptor Agonist Vasoconstrictors and Anti-Pain/InflammationAgents Therapeutic Therapeutic Most Acceptable Efficient PreferredPreferred Concentrations Concentrations Concentrations ConcentrationCompounds (nM) (nM) (nM) (nM) clonidine 0.002-200,000 0.01-50,0000.1-10,000 10-2,000 dexmedetomidine 0.002-200,000 0.01-50,000 0.1-10,00010-2,000 UK14304 0.002-200,000 0.01-50,000 0.1-10,000 10-2,000oxymetazoline 0.001-100,000 0.01-25,000 0.05-15,000   5-10,000 NS-490.002-200,000 0.01-50,000 0.1-10,000 10-2,000 AGN192172 0.005-100,000 0.1-25,000  1-5,000 10-1,000 AGN193080 0.005-100,000  0.1-25,000 1-5,000 10-1,000 AGN191103 0.002-200,000  0.1-25,000  1-5,000 10-1,000A-54741 0.002-200,000  0.1-50,000   1-10,000 10-2,000 BHT9200.003-200,000  0.3-50,000   3-30,000 30-5,000 BHT933 0.003-200,000 0.3-50,000   3-30,000 30-5,000

2. Serotonin Receptor Antagonists

Serotonin (5-HT) is thought to produce pain by stimulating serotonin₂(5-HT₂) and/or serotonin₃ (5-HT₃) receptors on nociceptive neurons inthe periphery. Most researchers agree that 5-HT₃ receptors on peripheralnociceptors mediate the immediate pain sensation produced by 5-HT(Richardson et al., 1985). In addition to inhibiting 5-HT-induced pain,5-HT₃ receptor antagonists, by inhibiting nociceptor activation, alsomay inhibit neurogenic inflammation. Barnes P. J., et al., Modulation ofNeurogenic Inflammation: Novel Approaches to Inflammatory Disease,Trends in Pharmacological Sciences 11, pp. 185-189 (1990). A study inrat ankle joints, however, claims the 5-HT₂ receptor is responsible fornociceptor activation by 5-HT. Grubb, B. D., et al., A Study of5-HT-Receptors Associated with Afferent Nerves Located in Normal andInflamed Rat Ankle Joints, Agents Actions 25, pp. 216-18 (1988).Therefore, activation of 5-HT₂ receptors also may play a role inperipheral pain and neurogenic inflammation.

One goal of the solution of the present invention is to block pain and amultitude of inflammatory processes. Thus, 5-HT₂ and 5-HT₃ receptorantagonists are both suitably used, either individually or together, inthe solution of the present invention, as shall be describedsubsequently. Amitriptyline (Elavil™) is a suitable 5-HT₂ receptorantagonist for use in the present invention. Amitriptyline has been usedclinically for numerous years as an anti-depressant, and is found tohave beneficial effects in certain chronic pain patients. Metoclopramide(Reglan™) is used clinically as an anti-emetic drug, but displaysmoderate affinity for the 5-HT₃ receptor and can inhibit the actions of5-HT at this receptor, possibly inhibiting the pain due to 5-HT releasefrom platelets. Thus, it also is suitable for use in the presentinvention.

Other suitable 5-HT₂ receptor antagonists include imipramine, trazodone,desipramine and ketanserin. Ketanserin has been used clinically for itsanti-hypertensive effects. Hedner, T., et al., Effects of a NewSerotonin Antagonist, Ketanserin, in Experimental and ClinicalHypertension, Am J of Hypertension, pp. 317s-23s (July 1988). Othersuitable 5-HT₃ receptor antagonists include cisapride and ondansetron.Solution also may contain a serotonin_(1B) (also known asserotonin_(1Dβ)) antagonist. Suitable serotonin_(1B) receptorantagonists include yohimbine,N-[-methoxy-3-(4-methyl-1-piperanzinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide(“GR127935”) and methiothepin. Therapeutic and preferred concentrationsfor use of these drugs in the solution of the present invention are setforth in Table 3.

TABLE 3 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Serotonin₂ Receptor Antagonists:Amitriptyline  100-50,000 1,000-25,000 imipramine 0.1-1,000 50-500trazodone 0.1-2,000 50-500 desipramine 0.1-1,000 50-500 ketanserin0.1-1,000 50-500 Serotonin₃ Receptor Antagonists: tropisetron 0.01-100  0.05-50   metoclopramide   10-10,000  200-2,000 cisapride 0.1-1,00020-200 ondansetron 0.1-1,000 20-200 Serotonin_(1B) (Human 1D_(β))Antagonists: yohimbine 0.1-1,000 50-500 GR127935 0.1-1,000 10-500methiothepin 0.1-500    1-100

3. Serotonin Receptor Agonists

5-HT_(1A), 5-HT_(1B) and 5-HT_(1D) receptors are known to inhibitadenylate cyclase activity. Thus including a low dose of theseserotonin_(1A), serotonin_(1B) and serotonin_(1D) receptor agonists inthe solution should inhibit neurons mediating pain and inflammation. Thesame action is expected from serotonin_(1E) and serotonin_(1F) receptoragonists because these receptors also inhibit adenylate cyclase.

Buspirone is a suitable 1A receptor agonist for use in the presentinvention. Sumatriptan is a suitable 1A, 1B, 1D and 1F receptor agonist.A suitable 1E receptor agonist is ergonovine. Therapeutic and preferredconcentrations for these receptor agonists are provided in Table 4.

TABLE 4 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Serotonin_(1A) Agonists:buspirone 1-1,000 10-200 sumatriptan 1-1,000 10-200 Serotonin_(1B)Agonists: sumatriptan 1-1,000 10-200 Serotonin_(1D) Agonists:sumatriptan 1-1,000 10-200 Serotonin_(1E) Agonists: ergonovine 10-2,000  100-1,000 Serotonin_(1F) Agonists: sumatriptan 1-1,000 10-200

4. Histamine Receptor Antagonists

Histamine receptors generally are divided into histamine₁ (H₁) andhistamine₂ (H₂) subtypes. The classic inflammatory response to theperipheral administration of histamine is mediated via the H₁ receptor.Douglas, 1985. Therefore, the solution of the present inventionpreferably includes a histamine H₁ receptor antagonist. Promethazine(Phenergan™) is a commonly used anti-emetic drug, which potently blocksH₁ receptors, and is suitable for use in the present invention.Interestingly, this drug also has been shown to possess local anestheticeffects but the concentrations necessary for this effect are severalorders higher than that necessary to block H₁ receptors, thus, theeffects are believed to occur by different mechanisms. The histaminereceptor antagonist concentration in the solution is sufficient toinhibit H₁ receptors involved in nociceptor activation, but not toachieve a “local anesthetic” effect, thereby eliminating the concernregarding systemic side effects.

Other suitable H₁ receptor antagonists include terfenadine,diphenhydramine, amitriptyline, mepyramine and tripolidine. Becauseamitriptyline is also effective as a serotonin₂ receptor antagonist, ithas a dual inflammation/pain inhibitory function as used in the presentinvention. Suitable therapeutic and preferred concentrations for each ofthese H₁ receptor antagonists are set forth in Table 5.

TABLE 5 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Histamine₁ Receptor Antagonists:promethazine 0.1-1,000 50-200 diphenhydramine 0.1-1,000 50-200amitriptyline 0.1-1,000 50-500 terfenadine 0.1-1,000 50-500 mepyramine(pyrilamine) 0.1-1,000  5-200 tripolidine 0.01-100   5-20

5. Bradykinin Receptor Antagonists

Bradykinin receptors generally are divided into bradykinin₁ (B₁) andbradykinin₂ (B₂) subtypes. Studies have shown that acute peripheral painand inflammation produced by bradykinin are mediated by the B₂ subtypewhereas bradykinin-induced pain in the setting of chronic inflammationis mediated via the B₁ subtype. Perkins, M. N., et al., AntinociceptiveActivity of the Bradykinin B1 and B2 Receptor Antagonists, des-Arg ⁹ ,[Leu ⁸]-BK and HOE 140, in Two Models of Persistent Hyperalgesia in theRat, Pain 53, pp. 191-97 (1993); Dray, A., et al., Bradykinin andInflammatory Pain, Trends Neurosci 16, pp. 99-104 (1993), each of whichreferences is hereby expressly incorporated by reference.

At present, bradykinin receptor antagonists are not used clinically.These drugs are peptides (small proteins), and thus they cannot be takenorally, because they would be digested. Antagonists to B₂ receptorsblock bradykinin-induced acute pain and inflammation. Dray et al., 1993.B₁ receptor antagonists inhibit pain in chronic inflammatory conditions.Perkins et al., 1993; Dray et al., 1993. Therefore, depending on theapplication, the solution of the present invention preferably includeseither or both bradykinin B₁ and B₂ receptor antagonists. For example,arthroscopy is performed for both acute and chronic conditions, and thusan irrigation solution for arthroscopy could include both B₁ and B₂receptor antagonists.

Suitable bradykinin receptor antagonists for use in the presentinvention include the following bradykinin₁ receptor antagonists: the[des-Arg¹⁰] derivative of D-Arg-(Hyp³-Thi⁵-D-Tic⁷-Oic⁸)-BK (“the[des-Arg¹⁰] derivative of HOE 140”, available from HoechstPharmaceuticals); and [Leu⁸] des-Arg⁹-BK. Suitable bradykinin₂ receptorantagonists include: [D-Phe⁷]-BK; D-Arg-(Hyp³-Thi^(5,8)-D-Phe⁷)-BK (“NPC349”); D-Arg-(Hyp³-D-Phe⁷)-BK (“NPC 567”); andD-Arg-(Hyp³-Thi⁵-D-Tic⁷-Oic⁸)-BK (“HOE 140”). These compounds are morefully described in the previously incorporated Perkins et al. 1993 andDray et al. 1993 references. Suitable therapeutic and preferredconcentrations are provided in Table 6.

TABLE 6 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Bradykinin₁ Receptor Antagonists:[Leu⁸] des-Arg⁹-BK 1-1,000 50-500 [des-Arg¹⁰] derivative of HOE 1401-1,000 50-500 [leu⁹] [des-Arg¹⁰] kalliden 0.1-500   10-200 Bradykinin₂Receptor Antagonists: [D-Phe⁷]-BK 100-10,000   200-5,000 NPC 349 1-1,00050-500 NPC 567 1-1,000 50-500 HOE 140 1-1,000 50-500

6. Kallikrein Inhibitors

The peptide bradykinin is an important mediator of pain andinflammation, as noted previously. Bradykinin is produced as a cleavageproduct by the action of kallikrein on high molecular weight kininogensin plasma. Therefore kallikrein inhibitors are believed to betherapeutic in inhibiting bradykinin production and resultant pain andinflammation. A suitable kallikrein inhibitor for use in the presentinvention is aprotinin. Suitable concentrations for use in the solutionsof the present invention are set forth below in Table 7.

TABLE 7 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Kallikrein Inhibitor: 0.1-1,00050-500 Aprotinin

7. Tachykinin Receptor Antagonists

Tachykinins (TKs) are a family of structurally related peptides thatinclude substance P, neurokinin A (NKA) and neurokinin B (NKB). Neuronsare the major source of TKs in the periphery. An important generaleffect of TKs is neuronal stimulation, but other effects includeendothelium-dependent vasodilation, plasma protein extravasation, mastcell recruitment and degranulation and stimulation of inflammatorycells. Maggi, C. A., Gen. Pharmacol., Vol. 22, pp. 1-24 (1991). Due tothe above combination of physiological actions mediated by activation ofTK receptors, targeting of TK receptors is a reasonable approach for thepromotion of analgesia and the treatment of neurogenic inflammation.

a. Neurokinin₁ Receptor Subtype Antagonists

Substance P activates the neurokinin receptor subtype referred to asNK₁. Substance P is an undecapeptide that is present in sensory nerveterminals. Substance P is known to have multiple actions, which produceinflammation and pain in the periphery after C-fiber activation,including vasodilation, plasma extravasation and degranulation of mastcells. Levine, J. D., et al., Peptides and the Primary AfferentNociceptor, J. Neurosci. 13, p. 2273 (1993). A suitable Substance Pantagonist is([D-Pro⁹[spiro-gamma-lactam]Leu¹⁰,Trp¹¹]physalaemin-(1-11)) (“GR82334”). Other suitable antagonists for use in the present inventionwhich act on the NK₁ receptor are:1-imino-2-(2-methoxy-phenyl)-ethyl)-7,7-diphenyl-4-perhydroisoindolone(3aR,7aR)(“RP 67580”); and2S,3S-cis-3-(2-methoxybenzylamino)-2-benzhydrylquinuclidine (“CP96,345”). Suitable concentrations for these agents are set forth inTable 8.

TABLE 8 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Neurokinin₁ Receptor SubtypeAntagonists GR 82334 1-1,000  10-500  CP 96,345 1-10,000 100-1,000 RP67580 0.1-1,000  100-1,000

b. Neurokinin₂ Receptor Subtype Antagonists

Neurokinin A is a peptide which is co-localized in sensory neurons withsubstance P and which also promotes inflammation and pain. Neurokinin Aactivates the specific neurokinin receptor referred to as NK₂.Edmonds-Alt, S., et al., A Potent and Selective Non-Peptide Antagonistof the Neurokinin A (NK ₂) Receptor, Life Sci. 50:PL101 (1992). Examplesof suitable NK₂ antagonists include:((S)-N-methyl-N-[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide(“(±)-SR 48968”); Met-Asp-Trp-Phe-Dap-Leu (“MEN 10,627”); andcyc(Gln-Trp-Phe-Gly-Leu-Met) (“L 659,877”). Suitable concentrations ofthese agents are provided in Table 9.

TABLE 9 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Neurokinin₂ Receptor SubtypeAntagonists: MEN 10,627 1-1,000 10-1,000 L 659,877 10-10,000 100-10,000(±)-SR 48968 10-10,000 100-10,000

8. CGRP Receptor Antagonists

Calcitonin gene-related peptide (CGRP) is a peptide which is alsoco-localized in sensory neurons with substance P, and which acts as avasodilator and potentiates the actions of substance P. Brain, S. D., etal., Inflammatory Oedema Induced by Synergism Between CalcitoninGene-Related Peptide (CGRP) and Mediators of Increased VascularPermeability, Br. J. Pharmacol. 99, p. 202 (1985). An example of asuitable CGRP receptor antagonist is α-CGRP-(8-37), a truncated versionof CGRP. This polypeptide inhibits the activation of CGRP receptors.Suitable concentrations for this agent are provided in Table 10.

TABLE 10 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) CGRP Receptor: 1-1,000 10-500Antagonist α-CGRP-(8-37)

9. Interleukin Receptor Antagonist

Interleukins are a family of peptides, classified as cytokines, producedby leukocytes and other cells in response to inflammatory mediators.Interleukins (IL) may be potent hyperalgesic agents peripherally.Ferriera, S. H., et al., Interleukin-1β as a Potent Hyperalgesic AgentAntagonized by a Tripeptide Analogue, Nature 334, p. 698 (1988). Anexample of a suitable IL-1β receptor antagonist is Lys-D-Pro-Thr, whichis a truncated version of IL-1β. This tripeptide inhibits the activationof IL-1β receptors. Suitable concentrations for this agent are providedin Table 11.

TABLE 11 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Interleukin Receptor 1-1,00010-500 Antagonist: Lys-D-Pro-Thr

10. Inhibitors of Enzymes Active in the Synthetic Pathway forArachidonic Acid Metabolites a. Phospholipase Inhibitors

The production of arachidonic acid by phospholipase A₂ (PLA₂) results ina cascade of reactions that produces numerous mediators of inflammation,know as eicosanoids. There are a number of stages throughout thispathway that can be inhibited, thereby decreasing the production ofthese inflammatory mediators. Examples of inhibition at these variousstages are given below.

Inhibition of the enzyme PLA₂ isoform inhibits the release ofarachidonic acid from cell membranes, and therefore inhibits theproduction of prostaglandins and leukotrienes resulting in decreasedinflammation and pain. Glaser, K. B., Regulation of Phospholipase A2Enzymes: Selective Inhibitors and Their Pharmacological Potential, Adv.Pharmacol. 32, p. 31 (1995). An example of a suitable PLA₂ isoforminhibitor is manoalide. Suitable concentrations for this agent areincluded in Table 12. Inhibition of the phospholipase C_(γ) (PLC_(γ))isoform also will result in decreased production of prostanoids andleukotrienes, and, therefore, will result in decreased pain andinflammation. An example of a PLC_(γ) isoform inhibitor is1-[(6-((17β-3-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

TABLE 12 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) PLA₂ Isoform 100-100,000500-10,000 Inhibitor: manoalide

b. Cyclooxygenase Inhibitors

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used asanti-inflammatory, anti-pyretic, anti-thrombotic and analgesic agents.Lewis, R. A., Prostaglandins and Leukotrienes, In: Textbook ofRheumatology, 3d ed. (Kelley W. N., et al., eds.), p. 258 (1989). Themolecular targets for these drugs are type I and type II cyclooxygenases(COX-1 and COX-2). These enzymes are also known as Prostaglandin HSynthase (PGHS)-1 (constitutive) and -2 (inducible), and catalyze theconversion of arachidonic acid to Prostaglandin H which is anintermediate in the biosynthesis of prostaglandins and thromboxanes. TheCOX-2 enzyme has been identified in endothelial cells, macrophages, andfibroblasts. This enzyme is induced by IL-1 and endotoxin, and itsexpression is upregulated at sites of inflammation. Constitutiveactivity of COX-1 and induced activity of COX-2 both lead to synthesisof prostaglandins which contribute to pain and inflammation.

i. Non-Selective Cyclooxygenase Inhibitors

NSAIDs currently on the market (diclofenac, naproxen, indomethacin,ibuprofen, etc.) are generally nonselective inhibitors of both isoformsof COX, but may show greater selectively for COX-1 over COX-2, althoughthis ratio varies for the different compounds. Use of COX-1 and 2inhibitors to block formation of prostaglandins represents a bettertherapeutic strategy than attempting to block interactions of thenatural ligands with the seven described subtypes of prostanoidreceptors. Reported antagonists of the eicosanoid receptors (EP-1, EP-2,EP-3) are quite rare and only specific, high affinity antagonists of thethromboxane A2 receptor have been reported. Wallace, J. and Cirino, G.Trends in Pharm. Sci., Vol. 15 pp. 405-406 (1994).

The oral, intravenous or intramuscular use of cyclooxygenase inhibitorsis contraindicated in patients with ulcer disease, gastritis or renalimpairment. In the United States, the only available injectable form ofthis class of drugs is ketorolac (Toradol™), available from SyntexPharmaceuticals, which is conventionally used intramuscularly orintravenously in postoperative patients but, again, is contraindicatedfor the above-mentioned categories of patients. The use of ketorolac, orany other cyclooxygenase inhibitor(s), in the solution in substantiallylower dosages than currently used perioperatively may allow the use ofthis drug in otherwise contraindicated patients. The addition of acyclooxygenase inhibitor to the solutions of the present invention addsa distinct mechanism for inhibiting the production of pain andinflammation during arthroscopy or other therapeutic or diagnosticprocedure.

Another cyclooxygenase inhibitor suitable for use in the presentinvention to inhibit pain and inflammation is ketoprofen. Ketoprofen isa non-selective COX inhibitor, and is also reported to have anadditional mechanism of action, namely the inhibition of thelipoxygenase pathway. Ketoprofen's action on COX-1 and COX-2 blocks theproduction of both prostaglandins and thromboxanes, and through actionon 5-lipoxygenase, it is also expected to inhibit formation ofleukotrienes and 5-HETE. Ketoprofen prevents the formation of both COXand lipoxygenase products (e.g., prostaglandins and leukotrienes,respectively) via inhibition of the release of arachidonic acid fromphospholipid membranes. This dual mechanism of action may havetherapeutic advantage, particularly in inflammatory pain states. Indeed,the efficacy of ketoprofen in a carrageenan model of inflammatory painwas greater than that obtained with other NSAIDs in the sameexperimental paradigm. Buritova, J., et al, Pain 6: 379-389 (1996);Honore P, et al, Pain 6: 365-375 (1995). Ketoprofen also exhibits highpotency characterized in cellular and animal models of jointneuroinflammation, rapid onset kinetics characterized in experimentalsystems, and an absence of effects on cartilage metabolism. Ketoprofen'slocal anti-inflammatory action due to reduced synthesis of vasodilatorprostaglandins (PGE2 and PGI2) also inhibits local vasodilatation andincreased capillary permeability associated with the acute inflammatoryresponse.

Preferred cyclooxygenase inhibitors for use in the present invention areketoprofen, keterolac and indomethacin. Of these agents, ketoprofen ismost preferred. Therapeutic and preferred concentrations for use in thesolution are provided in Table 13.

TABLE 13 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Cyclooxygenase Inhibitors:ketorolac  100-10,000  500-5,000 indomethacin 1,000-500,00010,000-200,000 Ketoprofen 1,000-500,000  5,000-100,000

ii. Cyclooxygenase-2 (COX-2) Inhibitors

As noted above, it is now appreciated that there are two forms ofcyclooxygenase, termed cyclooxygenase-1 or type 1 (COX-1) andcyclooxygenase-2 or type 2 (COX-2). These isozymes are also known asProstaglandin H Synthase (PGHS)-1 and PGHS-2, respectively. Both enzymescatalyze the conversion of arachidonic acid to unstable intermediates,PGG₂ and PGH₂, which are intermediates in the biosynthesis ofprostaglandins and thromboxanes. COX-1 is present in platelets andendothelial cells and exhibits constitutive activity. COX-2 has beenidentified in endothelial cells, macrophages and fibroblasts, includingsynovial cells after treatment (induction) with cytokines

The COX-2 isozyme is induced in settings of inflammation by cytokinesand inflammatory mediators, such as IL-1, TNF-α and endotoxin, and itsexpression is upregulated at sites of inflammation. The large increasein activity of COX-2 above basal COX-1 activity concomitant with itsupregulation, leads to synthesis of prostaglandins, which contribute topain and inflammation. Because COX-2 is usually expressed only ininflamed tissue or after exposure to mediators of inflammation,selective inhibitors may exhibit anti-inflammatory activity withoutsimultaneous effects on constitutively expressed COX-1 activity presentin platelets and other cell types. This is considered to be a cause ofundesirable side effects associated with certain useages of somenonselective NSAID drugs (e.g., clotting time, bleeding and ulceration).

It has been established that the two COX isozymes are pharmacologicallydistinct and therefore it has been possible to develop isozyme-specific(selective) cyclooxygenase inhibitors that are useful foranti-inflammatory therapy. A variety of biochemical, cellular and animalassays have been developed to assess the relative selectivity ofinhibitors for the COX-1 and COX-2 isoforms. These assays includemeasurements of prostaglandin E2 production in microsomes prepared fromvarious cell types and bioassay systems using intact human cells. Forany given drug, despite experimental variation in the degree ofselectivity noted among assay systems and between biological sources,compounds that are selective inhibitors for COX-2 have been identified.In general, a criteria for defining selectivity is the ratio of theCOX-1/COX-2 inhibitory constants (or COX-2/COX-1) obtained for a givenbiochemical or cellular assay system. The selectivity ratio accounts fordifferent absolute IC₅₀ values for inhibition of enzymatic activity thatare obtained between microsomal and cellular assay systems (e.g.platelets and macrophages, cell lines stably expressing recombinanthuman COX isozymes).

Many of the conventional NSAIDs currently on the market (naproxen,indomethacin, ibuprofen) are generally nonselective inhibitors of bothisoforms of COX, but may show greater selectively for COX-1 over COX-2,although this ratio varies for the different compounds. The use of aCOX-2 inhibitor to block formation of prostaglandins represents apreferred therapeutic strategy rather than attempting to blockinteractions of the endogenous prostanoid ligands, (such as PGE2, whichare produced by COX-2 at the inflammatory site, with any of the eightdescribed subtypes of prostanoid receptors. This approach is notcurrently feasible since selective and potent antagonists for all of theprostanoid receptors (EP-1, EP-2, EP-3, EP-4, DP, FP, IP and TP) do notexist.

A study by Riendeau and coworkers compared the selectivity of more than45 NSAIDs and selective COX-2 inhibitors using sensitive microsomal andplatelet assays for the inhibition of human COX-1 based on theproduction of prostaglandin E2 by microsomes (Can J. Physiol. Pharmacol(1997) 75:1088-95). In this study, among the compounds that werereported to show selectivity for COX-2 vs. COX1, the rank order ofpotency was DuP 697>SC-58451,celecoxib>nimesulide=meloxicam=piroxicam=NS-398=RS-57067>SC-57666>SC-58125>flosulide>etodolac>L-745337>DFU-T-614,with IC₅₀ values ranging from 7 μM to 17 μM. A good correlation wasobtained between the IC₅₀ values for the inhibition of microsomal COX-1and both the inhibition of TXB₂ production by Ca²+ionophore challengedplatelets and the inhibition of prostaglandin E2 production by CHO cellsstably expressing human COX-1. The microsomal assay was more sensitiveto inhibition than cell-based assays and allowed the detection ofinhibitory effects on COX-1 for all NSAIDs and selective COX-2inhibitors examined with discrimination of their potency underconditions of limited availability of arachidonic acid.

From the molecular and cellular mechanism of action defined forselective COX-2 inhibitors, such as celecoxib, as well as animalstudies, these compounds are expected to exhibit anti-inflammatoryaction when applied intraoperatively in an irrigation solution directlyto a tissue or a joint. In particular, it is expected to be an effectivedrug delivered by an irrigation solution during an arthroscopicprocedure (periprocedural).

Representative examples of suitable COX-2 inhibitors for use inconnection with the practice of the present invention include, withoutlimitation: celecoxib, meloxicam, nimesulide, nimesulide, diclofenac,flosulide, N42-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide(NS-398),1-[(4-methylsulfonyl)phenyl]-3-trifluoromethyl-5-[(4-fluoro)phenyl]pyrazole(SC58125), and the following compounds as described in Riendeau, D. etal., (1997) Can. J. Physiol. Pharmacol. 75: 1088-95: DuP 697, SC-58451,RS-57067, SC-57666 and L-745,337. Representative dosage levels foradministration in connection with the invention are listed in Table 14below.

TABLE 14 Cyclooxygenase-2 Inhibitors Therapeutic Most TherapeuticTherapeutic Preferred Preferred Acceptable Efficient Concen- Concen-Concentrations Concentrations trations trations Compounds (nM) (nM) (nM)(nM) DuP 697 0.01-50,000 0.05-15,000 0.3-3,000 3-500 SC-584510.01-50,000 0.05-15,000 0.3-3,000 3-500 celecoxib 0.01-50,0000.05-15,000 0.3-3,000 3-500 meloxicam 0.02-100,000  0.1-20,000 0.5-5,0005-1,000 nimesulide 0.02-100,000  0.1-20,000 0.5-5,000 5-1,000 diclofenac0.02-50,000  0.1-15,000 0.3-3,000 3-500 NS-398 0.01-50,000 0.06-15,0000.3-3,000 3-500 L-745,337 0.01-150,000 0.04-50,000 0.2-10,000 2-2,000RS57067 0.01-150,000 0.04-50,000 0.2-10,000 2-2,000 SC-581250.01-150,000 0.04-50,000 0.2-10,000 2-2,000 SC-57666 0.01-150,0000.04-50,000 0.2-10,000 2-2,000 flosulide 0.02-150,000 0.05-50,0000.2-10,000 2-2,000

c. Lipoxygenase Inhibitors

Inhibition of the enzyme lipoxygenase inhibits the production ofleukotrienes, such as leukotriene B₄, which is known to be an importantmediator of inflammation and pain. Lewis, R. A., Prostaglandins andLeukotrienes, In: Textbook of Rheumatology, 3d ed. (Kelley W. N., etal., eds.), p. 258 (1989). An example of a 5-lipoxygenase antagonist is2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (“AA861”), suitable concentrations for which are listed in Table 15.

TABLE 15 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Lipoxygenase Inhibitor:100-10,000 500-5,000 AA 861

11. Prostanoid Receptor Antagonists

Specific prostanoids produced as metabolites of arachidonic acid mediatetheir inflammatory effects through activation of prostanoid receptors.Examples of classes of specific prostanoid antagonists are theeicosanoid EP-1 and EP-4 receptor subtype antagonists and thethromboxane receptor subtype antagonists. A suitable prostaglandin E₂receptor antagonist is8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid,2-acetylhydrazide (“SC 19220”). A suitable thromboxane receptor subtypeantagonist is [15-[1α, 2β(5Z), 3β,4α]-7-[3-[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxobicyclo-[2,2,1]-hept-2-yl]-5-heptanoicacid (“SQ 29548”). Suitable concentrations for these agents are setforth in Table 16.

TABLE 16 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Eicosanoid EP-1 Antagonist:100-10,000 500-5,000 SC 19220

12. Leukotriene Receptor Antagonists

The leukotrienes (LTB₄, LTC₄, and LTD₄) are products of the5-lipoxygenase pathway of arachidonic acid metabolism that are generatedenzymatically and have important biological properties. Leukotrienes areimplicated in a number of pathological conditions includinginflammation. Specific antagonists are currently being sought by manypharmaceutical companies for potential therapeutic intervention in thesepathologies. Halushka, P. V., et al., Annu Rev. Pharmacol. Toxicol. 29:213-239 (1989); Ford-Hutchinson, A. Crit. Rev. Immunol. 10: 1-12 (1990).The LTB₄ receptor is found in certain immune cells including eosinophilsand neutrophils. LTB₄ binding to these receptors results in chemotaxisand lysosomal enzyme release thereby contributing to the process ofinflammation. The signal transduction process associated with activationof the LTB₄ receptor involves G-protein-mediated stimulation ofphosphotidylinositol (PI) metabolism and elevation of intracellularcalcium.

An example of a suitable leukotriene B₄ receptor antagonist is SC(+)-(S)-7-(3-(2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)-carbonyl]phenoxy(propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoicacid (“SC 53228”). Concentrations for this agent that are suitable forthe practice of the present invention are provided in Table 17. Othersuitable leukotriene B₄ receptor antagonists include[3-[-2(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino-3-oxopropyl)thio]methyl]thiopropanoicacid (“MK 0571”) and the drugs LY 66,071 and ICI 20,3219. MK 0571 alsoacts as a LTD₄ receptor subtype antagonist.

TABLE 17 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Leukotriene B₄ Antagonist:100-10,000 500-5,000 SC 53228

13. Opioid Receptor Agonists

Activation of opioid receptors results in anti-nociceptive effects and,therefore, agonists to these receptors are desirable. Opioid receptorsinclude the μ-, δ- and κ-opioid receptor subtypes. The μ-receptors arelocated on sensory neuron terminals in the periphery and activation ofthese receptors inhibits sensory neuron activity. Basbaum, A. I., etal., Opiate analgesia: How Central is a Peripheral Target?, N. Engl. J.Med., 325:1168 (1991). δ- and κ-receptors are located on sympatheticefferent terminals and inhibit the release of prostaglandins, therebyinhibiting pain and inflammation. Taiwo, Y. O., et al., Kappa-andDelta-Opioids Block Sympathetically Dependent Hyperalgesia, J.Neurosci., Vol. 11, page 928 (1991). The opioid receptor subtypes aremembers of the G-protein coupled receptor superfamily. Therefore, allopioid receptor agonists interact and initiate signaling through theircognate G-protein coupled receptor. Examples of suitable μ-opioidreceptor agonists are fentanyl and Try-D-Ala-Gly-[N-MePhe]-NH(CH₂)—OH(“DAMGO”). An example of a suitable δ-opioid receptor agonist is[D-Pen²,D-Pen⁵]enkephalin (“DPDPE”). An example of a suitable κ-opioidreceptor agonist is(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidnyl)cyclohexyl]-benzeneacetamide (“U50,488”). Suitable concentrations for each of these agentsare set forth in Table 18.

TABLE 18 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) μ-Opioid Agonist: DAMGO 0.1-1000.5-20   sufentanyl 0.01-50  1-20 fentanyl 0.1-500 10-200 PL 0170.05-50  0.25-10   δ-Opioid Agonist: DPDPE 0.1-500 1.0-100  κ-OpioidAgonist: U50,488 0.1-500 1.0-100 

14. Purinoceptor Antagonists and Agonists

Extracellular ATP acts as a signaling molecule through interactions withP₂ purinoceptors. One major class of purinoceptors are the P_(2x)purinoceptors which are ligand-gated ion channels possessing intrinsicion channels permeable to Na⁺, K⁺, and Ca²⁺. P_(2x) receptors describedin sensory neurons are important for primary afferent neurotransmissionand nociception. ATP is known to depolarize sensory neurons and plays arole in nociceptor activation since ATP released from damaged cellsstimulates P_(2X) receptors leading to depolarization of nociceptivenerve-fiber terminals. The P2X₃ receptor has a highly restricteddistribution (Chen, C. C., et al., Nature, Vol. 377, pp. 428-431 (1995))since it is selectively expressed in sensory C-fiber nerves that runinto the spinal cord and many of these C-fibers are known to carry thereceptors for painful stimuli. Thus, the highly restricted localizationof expression for the P2X₃ receptor subunits make these subtypesexcellent targets for analgesic action.

Suitable antagonists of P_(2X)/ATP purinoceptors for use in the presentinvention include, by way of example, suramin andpyridoxylphosphate-6-azophenyl-2,4-disulfonic acid (“PPADS”). Suitableconcentrations for these agents are provided in Table 19.

Agonists of the P_(2Y) receptor, a G-protein coupled receptor, are knownto effect smooth muscle relaxation through elevation of inositoltriphosphate (IP₃) levels with a subsequent increase in intracellularcalcium. An example of a P_(2Y) receptor agonist is 2-me-S-ATP.

TABLE 19 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Purinoceptor Antagonists: suramin100-100,000 10,000-100,000 PPADS 100-100,000 10,000-100,000

15. Adenosine Triphosphate (ATP)-Sensitive Potassium Channel Openers

ATP-sensitive potassium channels have been discovered in numeroustissues, including vascular and non-vascular smooth muscle and brain,and binding studies using radiolabeled ligands have confirmed theirexistence. Opening of these channels causes potassium (K⁺) efflux andhyperpolarizes the cell membrane. K⁺ channel openers (KCOs) have beenshown to prevent stimulus coupled secretion and are considered to act onprejunctional neuronal receptors and thus will inhibit effects due tonerve stimulation and release of inflammatory mediators. Quast, U., etal., Cellular Pharmacology of Potassium Channel Openers in VascularSmooth Muscle, Cardiovasc. Res., Vol. 28, pp. 805-810 (1994).

Suitable ATP-sensitive K⁺ channel openers for the practice of thepresent invention include: (−)pinacidil; cromakalim; nicorandil;minoxidil;N-cyano-N′-[1,1-dimethyl-[2,2,3,3-³H]propyl]-N″-(3-pyridinyl)guanidine(“P 1075”); and N-cyano-N′-(2-nitroxyethyl)-3-pyridinecarboximidamidemonomethansulphonate (“KRN 2391”). Concentrations for these agents areset forth in Table 20.

TABLE 20 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) ATP-Sensitive K⁺ Channel Opener:cromakalim 10-10,000 100-10,000 nicorandil 10-10,000 100-10,000minoxidil 10-10,000 100-10,000 P 1075 0.1-1,000   10-1,000 KRN 2391 1-10,000 100-1,000  (−)pinacidil  1-10,000 100-1,000 

16. MAP Kinase Inhibitors

The mitogen-activated protein (MAP) kinases are a group of proteinserine/threonine kinases that are activated in response to a variety ofextracellular stimuli and function in transducing signals from the cellsurface to the nucleus. The MAP kinase cascade is one of the majorsignaling pathways that transmit signals from growth factors, hormonesand inflammatory cytokines to intermediate early genes. In combinationwith other signaling pathways, these activated mitogen-activatedprotein-kinases (MAPKs) differentially alter the phosphorylation stateand activity of transcription factors, and ultimately regulate cellproliferation, differentiation and cellular response to environmentalstress. For example, MAPKs mediate the major signal transductionpathways from the potent inflammatory cytokine, IL-1, leading toinduction of cyclooxygenase-2 (COX-2) in stimulated macrophages, actingthrough cis-acting factors involved in the transcriptional regulation ofthe COX-2 gene.

Signaling from some G-protein-coupled receptors also involves the MAPKcascade, inducing a variety of responses including cell proliferation,differentiation, and activation of several intracellular kinasecascades. Prominent among these kinases are the activation of MAPkinases, including the extracellular signal-regulated kinases (ERKs),ERK1 and ERK2 (p44MAPK and p42MAPK, respectively); stress-activatedprotein kinases (SAPKs/JNKs); and p38 MAP kinase (also known asstress-activated kinase (SAPK)-2, reactivating kinase andcytokine-suppressive binding protein). These receptors signal throughheterotrimeric GTP-binding proteins (G-proteins). Recent data have shownthat the activation of mitogen-activated protein/ERK kinase induced byG-protein-coupled receptors is mediated by both Gα and Gβγ subunitsinvolving a common signaling pathway with receptor-tyrosine-kinases. Gβγmediated mitogen-activated protein kinase activation is mediated byactivation of phosphoinositide 3-kinase, followed by a tyrosinephosphorylation event, and proceeds in a sequence of events that involvefunctional association with the adaptor proteins Shc, Grb2, and Sos.Stress-activated protein kinases(SAPKs)/JNKs and p38 MAPK are able to beactivated by Gβγ proteins in a pathway involving Rho family proteinsincluding RhoA and Rac1.

A class of pyridinyl imidazoles inhibit p38 MAP kinase ((Lee, J. et al.(1994) Nature 372, 739-746)). Cuenda and coworkers (Cuenda, A. et al.,(1995) FEBS Lett. 364, 229) showed that the compound, SB203580[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]inhibited p38 in vitro (IC50=0.6 ìM), suppressed the activation ofMAPKAP kinase-2 and prevented the phosphorylation of heat shock protein(hsp) 27 in response to interleukin-1 (IL-1), cellular stresses andbacterial endotoxin in vivo. The specificity of SB203580 inhibitoryaction was demonstrated by its failure to inhibit 12 other proteinkinases in vitro (including ERKs). SB 203580 has become useful foridentifying the physiological roles and targets of p38 MAP kinase.

The role of p38 mitogen-activated protein kinase (MAPK) in biochemicalinflammatory responses of human fibroblasts and vascular endothelialcells to IL-1 was investigated by use of SB203580, which specificallyinhibits the enzyme. Actions of IL-1 that are selectively controlled byp38 MAPK are the regulation of prostaglandin H synthase-2 (also known asCOX-2), metalloproteinases, and IL-6 at different levels. (Ridley S H etal. (1997) J. Immunol. 158:3165-73). SB203580 inhibited (50% inhibitoryconcentration approximately 0.5 μM) IL-1-induced phosphorylation of hsp27 (an indicator of p38 MAPK activity) in fibroblasts without affectingthe other known IL-1-activated protein kinase pathways (p42/p44 MAPK,p54 MAPK/c-Jun N-terminal kinase). In addition, SB203580 significantlyinhibited IL-1-stimulated IL-6, (30 to 50% at 1 μM) but not IL-8production from human fibroblasts (gingival and dermal) and umbilicalvein endothelial cells. IL-1 induction of steady state level of IL-6mRNA was not significantly inhibited, which is consistent with p38 MAPKregulating IL-6 production at the translational level.

Importantly, SB203580 strongly inhibited IL-1-stimulated prostaglandinproduction by fibroblasts and human umbilical vein endothelial cells.This was associated with the inhibition of the induction of COX-2protein and mRNA. Since many cell types associated with inflammation,such as monocytes, endothelial cells and fibroblasts (includingsynovial) express the COX-2 gene at high levels upon activation bycytokines, extracellular stimuli and PGE2, the MAPK inhibitor isexpected to exhibit anti-inflammatory activity against all of thesecellular types Inhibitors of p38 MAP kinase are potent in inhibitingPGE2 release, which will result in anti-inflammatory benefits.

MAPK inhibitors may also be effective as cartilage protective agentswhen applied locally to tissues of the joint in a variety ofinflammatory or pathophysiological conditions. SB203580 was found toinhibit the stimulation of collagenase-1 and stromelysin-1 production byIL-1 without affecting synthesis of tissue inhibitor metalloproteinases(TIMP)-1. Furthermore, SB203580 prevented the increase in collagenase-1and stromelysin-1 mRNA stimulated by IL-1. In a model of cartilagebreakdown, short-term IL-1-stimulated proteoglycan resorption andinhibition of proteoglycan synthesis were unaffected by SB 203580, whilelonger term collagen breakdown was prevented.

p38 MAP kinase is involved in tumor necrosis factor (TNF)-inducedcytokine expression and drugs which function as inhibitors of p38 MAPkinase activity block the production of proinflammatory cytokines, asdescribed below (Beyaert, R. et al., EMBO J. 1996 15:1914-23). TNFtreatment of cells activated the p38 MAPK pathway, as revealed byincreased phosphorylation of p38 MAPK itself, activation of thesubstrate protein MAPKAP kinase-2, and phosphorylation of the heat shockprotein 27 (hsp27). Pretreatment of cells with the p38 MAP kinaseinhibitor SB203580 completely blocked this TNF-induced activation ofMAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions,SB203580 also completely inhibited TNF-induced synthesis of IL-6 andexpression of a reporter gene that was driven by a minimal promotercontaining two NF-Kappa B elements. Thus, these studies show that theaction of inhibitors, such as SB203580, on p38 MAPK interfereselectively with TNF-and IL-1 induced gene activation. SB 203580 hasbeen evaluated in several animal models of cytokine inhibition andinflammatory disease. It was demonstrated to be a potent inhibitor ofinflammatory cytokine production in vivo in both mice and rats with IC50values of 15 to 25 mg/kg. SB 203580 possessed therapeutic activity incollagen-induced arthritis in DBA/LACJ mice with a dose of 50 mg/kgresulting in significant inhibition of paw inflammation and serumamyloid protein levels. Antiarthritic activity was also observed inadjuvant-induced arthritis in the Lewis rat when SB203580 wasadministered p.o. at 30 and 60 mg/kg. Additional evidence was obtainedfor beneficial effects on bone resorption with an IC50 of 0.6 μM.

A large number of inflammatory mediators have been implicated inproducing synovitis of the joint, including arachidonic acid metabolites(particularly PGE2), vasoactive amines, and cytokines such as TNF-α,IL-1, IL-6 and neuropeptides. In fact, elevated levels of a number ofthese cytokines are found in the synovial fluid of acutely injured kneejoints and remain elevated in patients for at least 4 weeks. Thesecytokines are produced locally in the joint from several activated celltypes, including synovial fibroblasts, synovial macrophages, as well aschondrocytes.

In summary, a variety of biochemical, cellular and animal studies showthat p38 MAPK plays an important role in the regulation of responses toIL-1, TNF-α and LPS and it is involved in the regulation of mRNA levelsof some inflammatory-responsive genes, such as COX-2. Inhibitors of p38MAPK block the production of proinflammatory cytokines as well as PGE2and appear effective as anti-inflammatory drugs in animal models ofarthritis and bone resorption.

Pain and hyperalgesia commonly associated with inflammatory conditionsin the joint are in part due to activation of nociceptive sensoryneurons in the joint by PGE2 released as a result of the inflammatoryprocess. The ability of MAP kinase inhibitors to block the actions ofkey proinflammatory cytokines, such as IL-1 and TNF-α, will havedownstream effects on many cell types in the joint (synovial fibroblastsand chondrocytes) thus inhibiting subsequent pathological effects suchas infiltration of inflammatory cells into the joint, synovialhyperplasia, synovial cell activation, cartilage breakdown andinhibition of cartilage matrix synthesis. Thus, a MAPK inhibitor shouldblock the propagation of the pain and inflammatory response by theaforementioned cytokines, and thereby interrupt the disease process.

From the molecular and cellular mechanism of action defined for MAPkinase inhibitors, such as SB203580, these compounds are expected toexhibit anti-inflammatory action when applied intraoperatively in anirrigation solution directly to a tissue or a joint.

Representative examples of MAPK inhibitor compounds suitable for theinvention include, for example,4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole(SB203580),4-(3-Iodophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole(SB203580-iodo),4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole(SB202190),5-(2-amino-4-pyrimidyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole(SB220025),4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole (PD169316), and 2′-amino-3′-methoxyflavone (PD98059). Representative usefuldosages for these compounds are listed in the Table 21 below.

TABLE 21 MAP Kinase Inhibitors Therapeutic Most Therapeutic TherapeuticPreferred Preferred Acceptable Efficient Concen- Concen- ConcentrationsConcentrations trations trations Compounds (nM) (nM) (nM) (nM) SB 2035800.02-500,000  0.1-200,000 0.5-50,000 50-10,000 SB 203580 0.02-500,000 0.1-200,000 0.5-50,000 50-10,000 iodo SB 202190 0.01-500,0000.05-100,000 0.2-20,000 20-5,000 SB 220025 0.01-500,000 0.05-100,0000.2-25,000 20-5,000 PD 98059 0.01-500,000 0.04-50,000  0.1-10,00010-2,000 PD 169316 0.02-500,000  0.1-200,000   1-50,000 10-20,000

17. Neuronal Nicotinic Acetylcholine Receptor Agonists

Distinct receptor subtypes that comprise the nicotinic acetylcholinereceptor (nAChR) family are found on skeletal muscle at theneuromuscular junction, within the brain and spinal cord, on sensorynerves and some peripheral nerve terminals. These receptors function asligand-gated ion channels. Upon binding ligands that are agonists,nAChRs are transiently converted to an open channel state (activeconformation), which allows cation influx and subsequent depolarizationof the cell. Examples of ligands that function as agonists are thenatural neurotransmitter, acetylcholine, its nonhydrolyzable analog,carbamylcholine, DMPP, epibatidine and anatoxin-a. Antagonist ligandsinclude d-tubocurarine, and the snake venom α-neurotoxins, such asα-bungarotoxin. Invention compounds referred to as agonists include allligands which can be functionally classified as partial (weak andstrong) agonists and full agonists, thus encompassing the full spectrumof pharmacological agonist activity or efficacy based upon any method ofmeasurement, including electrophysiological responses measured byvoltage clamp technique, cellular or tissue based methods. Agonistsdefined by functional activity also include ligands that can act asallosteric modulators of neuronal nAChRs.

In the peripheral and central nervous systems, it is recognized thatthere is a molecular diversity of neuronal AChRs subtypes composed ofpentameric oligomers from a multi-gene family containing at least 13members (α₁-α₉, β₂-β₅). Molecular and biochemical approaches haveallowed neuronal nAChR subunits to be classified as either subunitsinvolved in binding of acetylcholine (α-subunits) or structural subunits(termed either as non-α or as β). The acetylcholine binding subunitshave been defined on the basis of adjacent cysteine residues (Cys 192and 193) in the primary sequences that are known to be part of theagonist binding site, and by reactivity with acetylcholine affinityalkylating agents. There are at least nine neuronal α-subunits (α₁-α₉)that can be divided into two classes on the basis of their ability tobind α-bungarotoxin (subunits α₇ and α₈) and at least four neuronal βsubunits (β₂-β₅).

A variety of functional neuronal nAChR subtypes have been constructed inheterologous expression studies. Pairwise coexpression of either α₂, α₃,or α₄ with β₂ or β₄ subunits has produced active acetylcholine-gated ionchannels. These expressed receptor subtypes differ in theirpharmacological profiles with respect to both agonist and antagonistsensitivities, as well as blockade by κ-bungarotoxin and are therebypharmacologically distinguishable. In contrast to other nAChR subunits,α₇ has been shown to form homooligomer receptors when expressed inXenopus_oocytes, and these active channels are characterized by highCa²⁺ conductance and rapid desensitization.

Clearly, there is a multitude of possible neuronal nAChR subtypevariations based upon combinations of five receptor subunits. Some ofthe pharmacological profiles for the expressed receptor subunitcombinations are correlated with properties of endogenously expressedreceptors found in ganglia, the CNS and in cell lines. nAChRs formed ofα₄ and β₂ subunits (nicotine binding sites) and α₇ (which bindα-bungarotoxin) represent the predominant subtypes in the mammalianbrain. Non-α₄ β₂ nAChRs have a more limited localization within the CNS.Receptor subtypes containing α₃ subunits are characteristic of humanganglionic nAChRs and are found in IMR-32 cells.

Evidence indicates neuronal nicotinic cholinergic channel agonists canfunction as potent analgesic agents by acting through neuronal nicotinicacetylcholine receptors (nAChRs). Recently, discovery of the potentantinociceptive actions of epibatidine have led to the identificationand development of novel neuronal nAChR subtype-selective nAChR ligandswith therapeutic potential as analgesic drugs. Substantial preclinicaland clinical data suggest that compounds that selectively activateneuronal nicotinic acetylcholine receptor subtypes will have therapeuticutility for the treatment of several neurological disorders, includingthe treatment of moderate and severe pain across a wide range ofconditions that include: acute, persistent inflammatory and neuropathicpain states. The specificity inherent in drugs targeted at neuronalreceptor subtypes allows for a defined mechanism of action with reducedside effect liabilities associated with interactions with nAChRs at theneuromuscular junction.

Abreo and coworkers (Abreo, M et al., (1996) J. Med. Chem 39:817-25)reported a novel series of 3-pyridyl ether compounds that possesssubnanomolar affinity for central neuronal nicotinic acetylcholinereceptors (nAChRs) and differentially activated subtypes of neuronalnAChRs. The synthesis and structure-activity relationships for theleading members of the series were described, including A-85380, whichpossesses a 50 pM affinity for rat brain [(3)H]-(−)-cytisine bindingsites and 163% efficacy compared to nicotine with regard to stimulationof ion flux at human α₄β₂ nAChR subtypes. In addition, A-84543 exhibited84-fold selectivity to stimulate ion flux at the human α₄β₂ nAChRsubtype compared to human ganglionic type nAChRs.

In another study, the in vitro pharmacological properties of a novelcholinergic channel modulator ABT-089[2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine], was described.Radioligand binding studies showed that ABT-089 displays selectivitytoward the high-affinity (−)-cytisine binding site present on the α₄β₂nAChR subtype (Ki=16 μM) relative to the [¹²⁵I]α-bungarotoxin bindingsite present on the neuronal α₇ subtype (Ki>10,000 μM) and the musclenAChR subtype of α₁β₁δ subunit composition (Ki>1000 μM).

The interaction of the nicotinic agonist(R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with differentnicotinic acetylcholine receptor (nAChR) subtypes has been establishedin studies employing cell lines and rat cortex. Zhang, X. et al.,Neurochem Int (1998) 32:435-41. In M10 cells, which stably express therecombinant α₄β₂ nAChR subtype, MPA showed an affinity (K_(i)=1.2 μM)which was higher thananatoxin-a>(−)-nicotine>(+)-[R]nornicotine>(−)-[S]nornicotine>and(+)-nicotine, but lower than cytisine (Ki=0.46 μM) in competing for(−)-[³H]nicotine binding. MPA showed a 13-fold higher affinity for(−)-[3H]nicotine binding sites compared to the [3H]epibatidine bindingsites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells,which predominantly express the endogenous α₃ nAChR subunit mRNA, MPAdisplaced [3H]epibatidine binding from sites with the same μM affinityas that observed in rat cortical membranes. MPA appears to have higherbinding affinity to the β4-subunit containing receptor subtype thanα₃-subunit containing receptor subtype. These studies furtherdemonstrate MPA binds to α₄β₂ receptor subtype with higher affinity than(−)-nicotine and behaves as a full agonist.

From the molecular and cellular mechanism of action defined for nAChRagonists, such as ABT-594, these compounds are expected to exhibitanti-nociceptive action on the peripheral terminals of primary afferentnerves when applied intraoperatively in an irrigation solution directlyto a tissue or a joint.

Representative examples of suitable neuronal nicotinic agonists for thepractice of the present invention include, without limitation:(R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594);(S)-5-(2-azetidinyl-methoxy)-2-chloropyridine (S-enatiomer of ABT-594);2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine (ABT-089);(R)-5-(2-Azetidinylmethoxy)-2-chloropyridine (ABT-594);(2,4)-Dimethoxy-benzylidene anabaseine (GTS-21); SBI-1765F and RJR-2403,as described in Holladay, M., Dart, M., and Lynch, J. (1997) J.Medicinal Chemistry 40:4169-4194;3-((1-methyl-2(S)-pyrrolidinyl)methoxy)pyridine (A-84543);3-(2(S)-azetidinylmethoxy)pyridine (A-85380); (+)-anatoxin-A and(−)anatoxin-A (1R)-1-(9-Azabicyclo[4.2.2]non-2-en-2-yl)-ethanoatefumarate, (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)azetidine (MPA), andothers shown below in Table 22.

TABLE 22 Neuronal Nicotinic Acetylcholine Receptor Agonists TherapeuticMost Acceptable Therapeutic Preferred Preferred Concen- EfficientConcen- Concen- trations Concentrations trations trations Compounds (nM)(nM) (nM) (nM) A-84543 0.01-250,000 0.02-50,000  0.1-10,000    10-2,000A-85380 0.02-500,000 0.1-100,000 1-20,000 100-4,000 ABT-089 0.02-500,0000.1-100,000 1-20,000 100-4,000 ABT-594 0.05-500,000 0.2-100,000 2-20,000 20-5,000 MPA 0.02-250,000 0.1-50,000  1-10,000  10-2,000 ABT-4180.02-500,000 0.1-100,000 1-20,000 100-5,000 GTS-21 0.02-500,0000.1-100,000 1-10,000 100-2,000 SIB-1765F 0.06-500,000 0.3-150,0003-15,000 300-6,000 RJR-2403 0.05-400,000 0.4-80,000  4-20,000  40-8,000cytisine 0.04-500,000 0.2-200,000 2-50,000  20-10,000 lobeline0.02-400,000 0.1-100,000 1-20,000  10-5,000

18. Soluble Receptors

Another class of agents useful in the present invention are solublereceptors. From a classical pharmacological perspective, the definitionof a receptor is based upon the concept that a receptor is able toselectively recognize a ligand and, importantly, provide a mechanism forthe transduction of this recognition event into a physiologicalresponse. At the cellular level, an operational definition of a receptoris that it must recognize a distinct ligand and transmit informationfrom the signal provided by the ligand into a form that alters the stateof the cell. Hence, the attributes of ligand recognition and signaltransduction are both used to define classes of receptors. Thetransduction process may be mediated through an integral part of thereceptor structure or may involve receptor interactions with additionalnon-receptor proteins (e.g. G-proteins), or some combination thereof.Receptor molecules belonging to the ligand-gated ion channel, G-proteincoupled, receptor tyrosine kinase, and cytokine superfamilies arelocated in the plasma membrane of the cell and mediate signaltransduction from a ligand bound to an extracellular ligand-bindingdomain of the receptor to an intracellular domain.

In contrast, soluble receptors retain the ability to selectivelyrecognize and bind their cognate ligands, but lack the capacity forsignal transduction. A number of endogenous soluble receptors areproduced and directly secreted by cells, or alternatively, are releasedfrom the extracellular membrane surface of cells into extracellularfluids. By the term “soluble,” it is intended that the receptorpolypeptide be soluble in aqueous solutions that include, but are notlimited to, detergent-free aqueous buffers, including saline andbuffered media, and body fluids such as extracellular fluid (ECF),blood, plasma and serum. While soluble receptors may be derived frommembrane-bound receptors, the uncomplexed soluble receptor is notanchored on cell surfaces. Specifically included are truncated orsoluble forms of the IL-1, IL-2, IL-4, IL-6, TNF, and FGF receptors nothaving a cytoplasmic and transmembrane region.

Within the context of defining soluble receptors as pharmacologicalantagonists, the term soluble receptor includes, but is not limited to:(1) soluble receptors which correspond to naturally (endogenous)produced amino acid sequences or soluble fragments thereof consisting ofan extracellular domain of a full-length membrane receptor; (2)recombinant soluble receptors which are truncated or partial amino acidsequences of the full-length naturally occurring receptor polypeptidewhich retain the ability to bind cognate ligand and retain biologicalactivity, and analogs thereof; and (3) chimeric soluble receptors whichare recombinant soluble receptors comprised of truncated or partialsequences corresponding to a portion of the extracellular binding domainof the full-length receptor amino acid sequences attached througholigomers (e.g. amino acids) to an amino acid sequence corresponding toa portion of an IgG polypeptide (e.g. IgG hinge and Fc domain) whichretain biological activity and the ability to bind cognate ligand.

Soluble, extracellular ligand-binding domains of cytokine receptorsoccur naturally in body fluids and are thought to be involved in theregulation of the biological activities of cytokines The naturallyoccurring existence of soluble, truncated forms of a number of cytokinereceptors has been reported (IL-1R, IL-4R, IL-6R, TNFR). For example,soluble TNFR is found at concentrations of about 1-2 ng/ml in the serumand urine of healthy subjects. Lacking signal transduction functions,these cytokine binding proteins arise as a result of alternativesplicing of the mRNA for the complete receptor sequence (membrane-boundform) or as a result of proteolytic cleavage and release of themembrane-bound form of the receptor. Although the in vivo functions ofthese soluble truncated receptors are not fully established, they appearto act as physiological antagonists of their complementary endogenouscytokines. This antagonism occurs because scavenging of the free ligandthrough binding to its cognate soluble receptor reduces the effectivefree concentration available to the membrane-bound receptors, andactions of the cytokines are only produced subsequent to binding to cellsurface receptors.

These soluble receptors can be viewed as natural antagonists of theircognate membrane-bound receptors by competing with cell surfacereceptors for common pool of free ligand. Thus, the pharmacologicalfunction of soluble receptors as antagonists is mediated by their uniqueability to alter free ligand bioavailability, rather than compete withan endogenous ligand for a common binding site on a membrane receptor.Addition of soluble receptors renders target cells less sensitive to theactivity of the cognate ligands, effectively neutralizing the biologicalactivity of the ligand. Experiments in which recombinant solublereceptors have been administered in vivo have demonstrated the capacityto inhibit inflammatory responses and act as antagonists.

Perioperative delivery of a soluble receptor(s), as defined herein, in aphysiologic carrier delivered directly to a surgical site, enables thesoluble receptor to act locally to reduce the levels of free or “active”endogenous polypeptides to preemptively inhibit inflammation and pain.

a. Classification and Examples of Soluble Receptors i. Tumor NecrosisFactor (TNF) Receptor Family

TNF-α is a cytokine mainly produced by activated macrophages that hasmany biological actions including cytotoxicity, anti-viral activity,immunoregulatory activities, and transcriptional regulation of severalgenes that are mediated by specific TNF receptors. Originally, twodifferent receptors termed TNF-R1 and TNF-R2 were cloned andcharacterized. Currently, 12 different TNF-related receptors have beenidentified (TNFR-1, TNFR-2, TNFR-RP, CD27, CD30, CD40, NGF receptor,PV-T2, PV-A53R, 4-1BB, OX-40, and Fas) with which eight differentTNF-related cytokines associate. All of these receptors (except PV-T2and PV-A53R) also exist as naturally produced, endogenous solublereceptors.

Receptors in this family are single transmembrane proteins withconsiderable homology in their extracellular domains whereas theirrelatively short intracellular domains bear very little sequencehomology. The actions of TNF are produced subsequent to binding of thefactor to cell surface receptors that are present on virtually all celltypes that have been studied. Two receptors have been identified andcloned. One receptor type, termed TNFR-II (or Type A or 75 kDa) shows anapparent molecular weight of 75 kDa. This gene encodes a transmembraneprotein of 439 amino acids. The other receptor type, termed TNFR-I (orType B or 55 kDa) shows an apparent molecular weight of 55 kDa andencodes a transmembrane protein of 426 amino acids. Both of thereceptors exhibit high affinity for binding TNFα. Soluble TNF receptors(sTNFR) have been isolated and proved to arise as a result of sheddingof the extracellular domains of the membrane-bound receptors. Two typesof sTNFR have been identified and designated as sTNFRI (TNF BPI) andsTNFRII (TNF BPII). Both of these soluble receptor forms have been shownto represent the truncated forms of the two types of TNFR describedabove.

TNFα plays a central role in the sequence of cellular and molecularevents underlying the inflammatory response. Among the proinflammatoryactions of TNF, it stimulates the release of other proinflammatorycytokines including IL-1, IL-6, and IL-8. TNFα also induces the releaseof matrix metalloproteinases from neutrophils, fibroblasts andchondrocytes. This cytokine, along with IL-1, is considered to initiateand produce pathological effects in the joint such as leukocyteinfiltration, synovial hyperplasia, synovial cell activation, cartilagebreakdown and inhibition of cartilage matrix synthesis. In particular,during acute inflammatory states, increased production of TNFα bysynovial cells occurs and increased levels of TNFα are found in thesynovial fluid of joints. Thus, local delivery of a soluble TNFαreceptor in an irrigation solution during a surgical procedure will bindfree TNFα and function as an antagonist of TNF receptors in thesurrounding tissue, thus providing an anti-inflammatory effect.

In one aspect, the present invention relates to the perioperativedelivery of a chimeric soluble receptor (CSR) protein, in which theextracellular domain of a TNF receptor (either TNFRI or TNRII), whichpossesses binding activity for a TNF molecule, is covalently linked to adomain of an IgG molecule. In particular, and by way of first example, achimeric polypeptide (recombinant chimera) comprising the extracellulardomain of the TNF receptor extracellular polypeptide coupled to the CH2and CH3 regions of a mouse IgG1 heavy chain polypeptide, as disclosed inU.S. Pat. No. 5,447,851, could be used for the present purpose. Thechimeric TNF soluble receptor (also termed the “chimeric TNF inhibitor”in U.S. Pat. No. 5,447,851) has been shown to bind TNFα with highaffinity and has been demonstrated to be highly active as an inhibitorof TNFα biological activity. In addition, a second example is a chimericfusion construct comprised of the ligand-binding domain of a TNFreceptor with portions of the Fc antibody (also termed Fc fusion solublereceptors) that have been created for TNFα receptors. In anotherembodiment, the present invention involves perioperative delivery of asoluble TNF receptor: Fc fusion protein, or modified forms thereof, asdisclosed in U.S. Pat. No. 5,605,690. The molecular form of the activesoluble receptor can be either monomeric or dimeric. Existing studiesestablish that such a soluble TNF receptor: Fc fusion protein (Enbrel)retains high binding affinity for TNFα and biological activity for TNFα.

ii. Interleukin-1 (IL-1) Cytokine Receptor Family

IL-1α and IL-1β are polypeptides that have a number of biologicalfunctions that include immunoregulatory, proinflammatory, andhematopoietic activities. A number of in vitro and in vivo experimentalstudies indicate that the ability to prevent the binding of IL-1 to itscell surface receptors will prevent IL-1 induced inflammatory andcartilage destructive effects within the joint. These actions aremediated by one of two IL-1 receptors (IL-1R), type I IL-1 (IL-1R1) ortype II IL-1 (IL-1 RII) receptors. The IL-1 receptors are structurallydistinct and belong to a separate superfamily characterized by thepresence of immunoglobulin-binding domains. The larger human type I IL-1receptor (80 kD) is present on numerous cell types, while the smallerhuman type II IL-1 receptor (60-68 kD) exhibits a more restricteddistribution that includes B cells, T-cells, monocytes, and neutrophils.Structurally, the human IL-1 R1 is a transmembrane glycoprotein with asubstantial intracellular domain composed of 213 amino acids (≅20 kD).The IL-1 RII receptor binds IL-1β with high affinity (about 2 nM), butIL-1β binding does not initiate IL-1 receptor associated intracellularsignal transduction as it does upon binding to the type I IL-1 receptor.Soluble receptor forms of both IL-1 R1 and IL-1 RII have been reported.The soluble form of IL-1 R1 is a 60 kD protein. The type II receptorserves as a precursor for a soluble IL-1 binding factor that is producedby proteolytic cleavage to yield two sizes of soluble receptors (47 kDand 57 kD).

A different type of naturally occurring, secreted soluble IL-1 receptorantagonist, alternatively referred to as the IL-1 antagonist protein(IL-1AP or IRAP) or the IL-1 receptor antagonist (IL-1RA or IL-1Ra), isexpressed in synovial tissue. It binds to both cell surface IL-1receptors, but does not induce any response and interacts with solubleIL-1 receptors. It is a product of several cell types found within thejoint, including synoviocytes and chondrocytes, as well as monocytes,macrophages and fibroblasts. This protein exists as two structuralvariant forms, characterized as a 17 kD secretory protein (sIL-1Ra) andan 18 kD form that remains in the cytoplasm. As a specific competitiveinhibitor of IL-1, IL-1Ra binds to the type I IL-1 receptor with highaffinity; it does not activate the cellular signal transductionmachinery activated by membrane associated IL-1 receptors. Soluble IL-1R1 also binds the IL-1 Ra with very high affinity (Kd=70 pM). Thesoluble type II receptor exhibits different binding characteristic thanthe membrane form of the receptors, exhibiting over 2000-fold loweraffinity for IL-1Ra. This results in IL-Ra having greater ability toantagonize IL-1 actions. The IL-1Ra has been shown to play aphysiological role in suppressing the biological actions of IL-1.Secreted IL-Ra is released in vivo during experimentally inducedinflammation and as part of the natural course of many diseases.

Agents useful in the present invention include an IL-1 soluble receptorprotein, which is formed of an extracellular domain of an IL-1R (eithertype I or II), and which is capable of binding an IL-1 cytokinemolecule. In particular, and by way of example, a soluble human IL-1receptor (shuIL-1R) polypeptide comprising essentially the amino acidsequence 1-312 as disclosed within U.S. Pat. No. 5,319,071 and U.S. Pat.No. 5,726,148 may be used in the present irrigation solutions.Alternatively, a fusion protein consisting of the sIL-1R binding domainpolypeptide, as disclosed in U.S. Pat. No. 5,319,071 may be used in theinvention. In addition, an IL-1 receptor antagonist as disclosed withinU.S. Pat. No. 5,817,306 can be employed for the present purpose. TheshuIL-1R soluble receptor has been shown to bind IL-1 with nanomolaraffinity. Local delivery of an IL-1R soluble receptor, such as shuIL-1R,in an irrigation solution at a therapeutically effective concentrationduring an arthroscopic procedure may be used as a cartilage protectiveagent when applied locally to tissues of the joint in a variety ofinflammatory or pathophysiological conditions. Such treatment willpreemptively inhibit IL-1 stimulation of production of collagenase-1 andstromelysin-1. Employing a wholly different method for local productionof type 1 soluble receptors for IL-1 and/or TNFα based on gene delivery,it has been found that the presence of soluble receptors for thesecytokines are able to confer protection to the rabbit knee joint duringthe acute inflammatory phase of a.i.a.

iii. Class I Cytokine Receptor Family

The large hematopoietic cytokine receptor superfamily consists of EPO,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15,Epo, PRL, GH, G-CSF, and GM-CSF, LIF, CNTF, and thrombopoietinreceptors. In general, these receptors mediate hematopoieticcytokine-induced growth and differentiation of hematopoietic cells, butalso exhibit a wide range of biological effects on various tissues andcells.

The receptor binding subunits for Class I cytokine receptors have beencharacterized at the molecular level and comparison of amino acidsequences has revealed several shared structural features or regions ofsequence homology. Class I cytokine receptors are characterized by thepresence of one or two copies of a conserved domain of about 200 aminoacids, which contain two modules of FN-III-like motifs located in theextracellular portion of the receptor. A second region is characterizedby a conserved cysteine motif (four conserved cysteines and onetryptophan residue) in the N-terminal half of this homology region. Alsocontained in this homology region is a common Trp-Ser-X-Trp-Ser sequence(where X is a nonconserved amino acid) at the C-terminal end. There arealso regions of shared amino acid sequence homology in the intracellulardomains of hematopoietic receptors that are referred to as homologyboxes 1 and 2.

The Class I cytokine receptor family has been subdivided into fourreceptor subfamilies based on common mechanisms of signal transductionresulting from cytokine binding to the receptor binding subunit. TheEPO, G-CSF, PRL and GH receptors comprise the GH receptor subfamily inwhich cytokine binding to a single receptor-binding subunit promotes theformation of a functional high affinity receptor dimer on the plasmamembrane. The other three subfamilies of hematopoietic receptors do notform dimers upon cytokine binding. Agonist binding to structurallyunique cytokine-binding subunits for each of the members in thesefamilies of hematopoietic cytokine receptors results in the formation ofa high-affinity complex with a shared signal transducing subunit.

Among the members of the Class I cytokine receptor family, solublereceptors for IL-2, IL-4, IL-6, and GM-CSF ligands are suitable forinclusion in irrigation solutions of the current invention. The use ofsoluble receptors for IL-6 and GM-CSF ligands is preferred inarthroscopic surgical procedures.

(a). Soluble IL-2 Receptor and IL-4 Receptor: IL-2R Subfamily

Within the IL-2 receptor (IL-2R) subfamily, the structurally uniquebinding subunits for IL-2, IL-4, IL-7, IL-9, IL-13 and IL-15 receptorsall form high-affinity functional complexes with a common signaltransduction protein referred to as the IL-2γ subunit. The IL-2receptor, unlike other receptors in this IL-2γ family, also associateswith a third transmembrane protein subunit, IL-2R13 (75 kD). In thiscase, the high affinity IL-2 receptor exists as a heterotrimericcomplex. A soluble form of the IL-2Rα receptor appears in serum,concomitant with its increased expression on cells and there are reportsof a soluble form of the IL-2Rβ. Agents useful in the solutions of thepresent invention include human IL-2Rβ soluble receptor (shIL-2R)proteins, in which the extracellular domain of a cytokine receptorpossesses binding activity for the IL-2 cytokine molecule. Inparticular, and by way of example, the human IL-2Rβ soluble receptor(shIL-2R) protein is disclosed within U.S. Pat. No. 5,449,756.

The ligand binding subunit of the human IL-4R present on the cellsurface receptor is a 140 kDa transmembrane glycoprotein containing 800amino acids: a 207 residue extracellular domain; a 24 residuetransmembrane domain; and a 569 residue intracellular domain. Inaddition to the full-length receptor, an alternatively spliced IL-4Rtranscript that encodes a secreted form of the IL-4R lacking thetransmembrane and cytoplasmic domains has been isolated from mousecells. A naturally occurring soluble form of the IL-4R has beenidentified in mouse biological fluids and murine cell culturesupernatants, as well as human serum. In solution, soluble IL-4R canform 1:1:1 complexes with IL-4 and the IL-2γ subunit.

(b). Soluble IL-6 Receptor: IL-6 Cytokine Receptor Subfamily

The IL-6 receptor subfamily of Class I cytokine receptors includes theIL-6, IL-11, CNTF, OSM, and LIF receptors. These all form ahigh-affinity functional receptor complex upon interaction with acytokine-occupied binding subunit with a common signal transductionprotein called gp130. In the case of the IL-6 receptor, IL-6 binding toits binding subunit leads to association with a homodimer of gp130instead of a single gp130 monomer. Recently, a human IL-12 bindingreceptor component has been cloned and found to be highly related inprimary structure to gp130.

There exist naturally occurring soluble forms of the IL-6R that bindIL-6 with high affinity. Soluble forms have also been identified inhuman serum as well as in the conditioned medium of various cells,including human peripheral mononuclear cells and T-cell lines. Elevatedserum soluble IL-6R levels have been shown to be associated with anumber of pathological states, including significant increases inpatients undergoing minor elective operations during the firstpostoperative week. Both soluble IL-6R and the soluble gp130 are presentin nanogram quantities in the serum of normal individuals. Although theexact mechanism generating sIL-6R is not understood, it has beendemonstrated that a naturally occurring alternate form of IL-6R mRNAexists and appears to be generated as a result of alternative splicingof mRNA, which encodes a soluble form of IL-6R lacking the transmembranedomain.

The various activities of IL-6 indicate it has a major role in themediation of the inflammatory response initiated by injury. IL-6 can beconsidered a critical proinflammatory cytokine, which is itselfupregulated in response to TNFα and IL-1 in a variety of disease statesand conditions. One of the major intraarticular cytokines that has beenstudied in the context of joint inflammation is IL-6. In a study ofcomparing changes in IL-6 levels in synovial fluid after anteriorcruciate ligament rupture in the knee, IL-6 increased about 1,500-foldin the acute injured knee. Thus, IL-6 is a target for the pharmacologiccontrol of inflammation. The present invention includes theperioperative delivery of a sIL-6R in an irrigation solution during anarthroscopic procedure in order to inhibit inflammation. The inhibitionof the proinflammatory activity of IL-6 by the IL-6 soluble receptor isof benefit in controlling or reducing inflammation in the joint.

iv. Receptor Tyrosine Kinases

A wide variety of polypeptide growth factor receptors that possessintrinsic tyrosine kinase activity have now been characterized for whichsoluble receptors are disclosed for use in the solutions of the presentinvention. Such agents include extracellular portions of receptortyrosine kinase receptors and chimeric soluble tyrosine kinasereceptors. Activated receptor tyrosine kinases (RTKs) undergodimerization and initiate signaling through tyrosine-specificphosphorylation of diverse intermediates, activating a cascade ofintracellular pathways that regulate phospholipid and arachidonatemetabolism, calcium mobilization, protein phosphorylation (involvingother protein kinases), and transcriptional regulation. Thegrowth-factor-dependent tyrosine kinase activity of the RTK cytoplasmicdomain is the primary mechanism for generation of intracellular signalsthat initiate multiple cellular responses.

Many of the RTK subfamilies are recognizable on the basis ofarchitectural similarities in the catalytic domain as well asdistinctive motifs in the extracellular ligand-binding regions. Theextracellular domain of the RTKs typically contains discrete structuralunits that are derived from a limited group of biochemical domains.These domains include: cysteine rich regions, immunoglobulin-like loops(IgLs), or fibronectin type III (FN-III) domains. Based upon thesestructural considerations, a nomenclature defining several subfamiliesof RTKs has been proposed. The eight receptor families referred to onthe basis of their prototypic members include: EGF-receptor, insulinreceptor, PDGF-receptor, the fibroblast growth factor receptor (FGFR),Neurotrophin (Trk) receptor, Hepatocyte Growth factor (HGF) receptor,Vascular Endothelial Growth Factor (VEGF) receptor and Eph receptors.Members of a given subfamily share common structural features that aredistinct from those found in other subfamilies.

A common structural feature shared by a group of three subfamilies, theEGF (EGFR, ErbB2, ErbB3, ErbB4), insulin and Eph (Eph, Elk, Eck, Cck5,Sek, Eck, and Erk) receptors, is the presence of cysteine-rich regionsin the extracellular domain. The Eph receptor extracellular region ischaracterized by a single cysteine-rich box that is related to the twotandem cysteine-rich boxes found in members of the EGF receptorsubfamily. Also containing a single cysteine-rich region, the insulinreceptor is the prototypic receptor for a subfamily whose distinctivestructural feature is its organization as a heterotetrameric species oftwo α and two β subunits. The extracellular ligand-binding subunit, α,is disulfide-linked to the transmembrane β subunit, which contains thetyrosine kinase domain.

A second major structural category is represented by a group of threesubfamilies, fibroblast-growth factor receptors (FGFR), platelet-derivedgrowth factor receptors (PDGFR) and Flt1/VEGF receptors, which arecharacterized by extracellular domains consisting of three, five, orseven IgLs. Cytoplasmic regions of these receptors contain a tyrosinekinase domain that is interrupted by a “kinase insert.” Receptorscontaining five IgLs include two PDGF receptors (α and β), themacrophage colony stimulating factor-1 receptor (CSF-1R), the c-kitprotein (a receptor for the steel ligand) and the product of theFLT3/FLK2 gene. The FGF receptors, which have three IgLs, constitute aseparate subfamily. Currently, there are at least seven FGFR membersthat mediate a diverse array of biological responses, including thecapacity to induce angiogenesis (FGFR-1, FGFR-2, FGFR-3 and FGFR-4). Inaddition, a group of RTKs with seven extracellular IgLs has beenproposed to represent a separate VEGF receptor subfamily. Its knownmembers, FLT1, FLK1 and FLT4, show a similarity of structure andexpression. Several lines of evidence suggest that this subfamily ofgrowth factor receptors play an important role in the growth anddifferentiation of endothelial cells.

One group of receptors that does not fall into either of the abovecategories is the Trk subfamily. Recent work on the Trk subfamily hasestablished that these molecules constitute signal-transducing receptorsfor a family of structurally and functionally related neurotrophicfactors, collectively known as the neurotrophins. This receptorsubfamily (Trk, TrkB, TrkC) contains neither cysteine-rich regions norIgLs in the extracellular domain. Instead, cysteines are foundthroughout the binding domain and are also clustered near theN-terminus.

Although there is a tremendous diversity among the numerous members ofthe RTK family, the signaling mechanisms used by these receptors sharemany common features. Biochemical and molecular genetic studies haveshown that binding of the ligand to the extracellular domain of the RTKrapidly activates the intrinsic tyrosine kinase catalytic activity ofthe intracellular domain which is essential for signal transduction.

For example, recombinant chimeric soluble receptors derived from Axl,Sky, Mer and c-Met (receptor for hepatocyte growth factor) and composedof the extracellular ligand-binding domain of these receptors fused tothe Fc region of the human immunoglobulin domain IgG1 heavy chain havebeen created (Nagata, et al., J. Biol. Chem., 271:30022-27, 1996).Naturally occurring counterparts for these chimeric receptors are notknown to exist. These tyrosine kinase receptor-Fc fusion (RTK-Fc)proteins were expressed in COS-7 cells and subsequently purified fromthe conditioned media by conventional protein A-Sepharosechromatography. Analysis showed these RTK-Fc fusion proteins wereexpressed as disulfide-linked dimers as has been found previously forother IgG fusion proteins. Binding analysis of the immobilized RTK-Fcfusion protein showed that the kinetics of specific protein ligandscould be quantitatively determined for Axl-Fc, Sky-Fc, and Mer-Fcsoluble receptors. This study confirmed that such chimeric solublereceptors retain binding affinity for endogenous protein ligands thatactivate the full-length endogenous forms of these receptors. Thus, inone aspect, the present invention is directed to the local delivery ofsuch RTK-Fc fusion proteins (or modified forms thereof) in an irrigationsolution as therapeutic agents to reduce the “active” form orconcentration of their respective cognate biological ligand.

Representative soluble receptors for use in the solutions of the presentinvention, and dosages, are listed in Table 23 below.

TABLE 23 Soluble Receptors Therapeutic Therapeutic Most TherapeuticPreferred Preferred Preferred Preferred Concen- Concen- Concen- SolubleConcentrations trations trations trations Receptor (nM) (nM) (nM) (nM)sTNFR 0.005-50,000  0.02-10,000 0.1-1000 1-200 Chimeric 0.005-50,000 0.02-10,000 0.1-1000 1-200 rhTNFR:Fc Human type I 0.01-50,0000.02-10,000 0.1-1000 1-200 IL-1R Human type II 0.01-50,000 0.02-10,0000.1-1000 1-200 IL-1R Shuman IL-1R 0.01-50,000 0.02-10,000 0.1-1000 1-200fusion protein with DYKDDDDK on N-terminus sIL-6R  0.01-100,0000.02-20,000 0.1-1000 1-200 bFGF receptor 0.01-50,000 0.02-5,000 0.1-1000 1-200 PDGF 0.01-50,000 0.02-5,000  0.1-1000 1-200

VI. METHOD OF APPLICATION

The solution of the present invention has applications for a variety ofoperative/interventional procedures, including surgical, diagnostic andtherapeutic techniques. The irrigation solution is perioperativelyapplied during arthroscopic surgery of anatomic joints. As used herein,the term “perioperative” encompasses application intraprocedurally, pre-and intraprocedurally, intra- and postprocedurally, and pre-, intra- andpostprocedurally. Preferably the solution is applied preprocedurallyand/or postprocedurally as well as intraprocedurally. Such proceduresconventionally utilize physiologic irrigation fluids, such as normalsaline or lactated Ringer's, applied to the surgical site by techniqueswell known to those of ordinary skill in the art. The method of thepresent invention involves substituting the anti-pain/anti-inflammatoryirrigation solutions of the present invention for conventionally appliedirrigation fluids. The irrigation solution is preferably applied to thewound or surgical site prior to the initiation of the procedure,preferably before tissue trauma, and continuously throughout theduration of the procedure, to preemptively block pain and inflammation.As used herein throughout, the term “irrigation” is intended to mean theflushing of a wound or anatomic structure with a stream of liquid. Theterm “application” is intended to encompass irrigation and other methodsof locally introducing the solution of the present invention, such asintroducing a gellable version of the solution to the operative site,with the gelled solution then remaining at the site throughout theprocedure. As used herein throughout, the term “continuously” isintended to also include situations in which there is repeated andfrequent irrigation of wounds at a frequency sufficient to maintain apredetermined therapeutic local concentration of the applied agents, andapplications in which there may be intermittent cessation of irrigationfluid flow necessitated by operating technique.

In addition to use during arthroscopic procedures, the solutions of theinvention may also be locally and perioperatively delivered during opensurgical procedures on joints of the extremities, including but notlimited to total knee, hip, ankle, toe, shoulder, elbow, wrist andfinger joint replacements, the placement of implants into joints of theextremities, and for other surgical procedures on an extremity. As usedherein, “extremity” refers to anatomic structures of the leg, includingthe hip, or of the arm, including the shoulder. Irrigation of opensurgical sites at joints or extremities may be carried out in accordancewith the invention by periodic direct irrigation with a bulb syringe orusing other conventional techniques.

As noted above, perioperative delivery of the solutions of the presentinvention during surgical procedures is preferred for a preemptive painand/or inflammation inhibitory effect. Solutions of the inventionincluding an alpha-selective adrenergic receptor agonist and one or moreadditional analgesic or anti-inflammatory agents in a physiologicirrigation carrier may also be used for direct irrigation of woundsbefore (preoperative) and/or during (intraoperative) and/or after(postoperative) an arthroscopic procedure, an open procedure on anextremity joint, or other surgical/interventional procedure on anextremity.

In a still further aspect of the invention, solutions of the inventionincluding an alpha-selective adrenergic receptor agonist and one or moreadditional analgesic or anti-inflammatory agents in a physiologiccarrier may be administered by intraarticular or intracapsular injectionof joints. Such injectable solutions may include a sustained releasevehicle for extended therapeutic effect.

The agents of the present invention may be delivered in a formulationuseful for introduction and administration of the drug into the targetedtissue or joint that enhances the delivery, uptake, stability orpharmacokinetics of the pharmacological agent. Suitable formulationsinclude, but are not limited to, administration using microparticles,microspheres or nanoparticles composed of lipids, proteins,carbohydrates, synthetic organic compounds, or inorganic compounds.Examples of formulation molecules include, but are not limited to,lipids capable of forming liposomes or other ordered lipid structures,cationic lipids, hydrophilic polymers such as poly (D,L lacticacid-coglycolic acid) polymers, chitosan, heparin, lipids capable offorming ordered lipid structures such as unilamellar and multilamellarliposomes (anionic, cationic and zwitterionic), polycations (e.g.protamine, spermidine, polylysine), peptide or synthetic ligands andantibodies capable of targeting materials to specific cell types, gelsincluding hydrogels, slow or sustained release matrices, soluble andinsoluble particles, as well as formulation elements not listed. Severalof these formulations utilize sustained release vehicles, includingmicroparticles, microspheres, nanoparticles, proteins, liposomes,carbohydrates, gels, and other slow or sustained release matrices.

The solutions of the present invention may be prepared at concentrationsgreater than that intended for local delivery. Such concentrates aresubsequently diluted with physiologic carrier to the desired localconcentration in accordance with the present invention, prior todelivery. The solutions of the present invention may also be prepared aslyophilized formulations, which are subsequently solubilized orreconstituted in a physiologic carrier at the desired concentrationprior to local delivery. The solutions of the present invention may alsoinclude excipients, such as stabilizers and buffers, for example.

In one aspect, the present invention provides for the local delivery ofthe pharmacological agents of the invention using an irrigation solutioncontaining the drug which is present at low concentration and whichenables the drug to be delivered directly to the affected tissue orjoint. The drug-containing irrigation solution is employedperioperatively during a surgical procedure. Other conventional methodsused for drug delivery have required systemic administration(intramuscular, intravenous, subcutaneous) which necessitates highconcentrations of drugs (and higher total dose) to be administered inorder to achieve significant therapeutic concentrations in the targetedtissue or joint (e.g., the synovial fluid of the joint). Systemicadministration also results in high concentrations in tissues other thanthe targeted tissue that is undesirable and, depending on the dose, mayresult in adverse side effects (e.g., bleeding, ulceration). Thesesystemic methods subject the drug to second pass metabolism and rapiddegradation, thereby limiting the duration of the effective therapeuticconcentration. Since the drug is administered directly to the desiredtissue, it does not depend upon vascular perfusion to carry the drug tothe targeted tissue. This significant advantage allows for the deliveryof the pharmacological agents of the invention using a therapeuticallyeffective lower concentration and lower therapeutically effective totaldose.

The concentrations listed for each of the agents within the solutions ofthe present invention are the concentrations of the agents deliveredlocally, in the absence of metabolic transformation, to the operativesite in order to achieve a predetermined level of effect at theoperative site. It is understood that the drug concentrations in a givensolution may need to be adjusted to account for local dilution upondelivery. Solution concentrations are not adjusted to account formetabolic transformations or dilution by total body distribution becausethese circumstances are avoided by local delivery, as opposed to oral,intravenous, subcutaneous or intramuscular application.

Arthroscopic techniques for which the present solution may be employedinclude, by way of non-limiting example, partial meniscectomies andligament reconstructions in the knee, shoulder acromioplasties, rotatorcuff debridements, elbow synovectomies, and wrist and anklearthroscopies. The irrigation solution is continuously suppliedintraoperatively to the joint at a flow rate sufficient to distend thejoint capsule, to remove operative debris, and to enable unobstructedintra-articular visualization.

A preferred solution for use in the present invention includes (a) acyclooxygenase inhibitor (most preferably a nonselective cyclooxygenaseinhibitor that also acts to inhibit lipoxygenase), (b) a serotonin₂antagonist and/or a histamine₁ antagonist (most preferably an agent thatexhibits both of these functions) and (c) an alpha adrenergic receptoragonist as a peripheral vasoconstrictor (more preferably an alphaagonist that is highly selective for alpha receptors without substantial(relatively little or no) interaction with beta receptors, and mostpreferably that is a mixed alpha-1 and alpha-2 agonist). One suchsuitable irrigation solution for control of pain and inflammation duringsuch arthroscopic techniques is provided in Example I herein below, andutilizes ketoprofen, amitriptyline and oxymetazoline for each of theagents listed above, respectively.

This solution utilizes extremely low doses of these pain andinflammation inhibitors, due to the local application of the agentsdirectly to the operative site during the procedure. For example, lessthan 0.05 mg of amitriptyline (a suitable serotonin₂ and histamine₁“dual” receptor antagonist) are needed per liter of irrigation fluid toprovide the desired effective local tissue concentrations that wouldinhibit 5-HT₂ and H₁ receptors. This dosage is extremely low relative tothe 10-25 mg of oral amitriptyline that is the usual starting dose forthis drug. This same rationale applies to other agents utilized in thesolutions of the present invention.

In each of the surgical solutions of the present invention, the agentsare included in low concentrations and are delivered locally in lowdoses relative to concentrations and doses required with conventionalmethods of drug administration to achieve the desired therapeuticeffect. It is impossible to obtain an equivalent therapeutic effect bydelivering similarly dosed agents via other (i.e., intravenous,subcutaneous, intramuscular or oral) routes of drug administration sincedrugs given systemically are subject to first- and second-passmetabolism. The agents are delivered locally in accordance with thepresent invention to provide a desired local level of therapeutic effectand results in a blood plasma level for the agent that is significantlyless than that which would result form systemic administration of theagent to achieve the same level of therapeutic effect. Given that only asmall fraction of the drug delivered intra-articularly is absorbed bythe local synovial tissue, the difference in plasma drug levels betweenthe two routes of administration is much greater than the difference intotal drug dosing levels.

Practice of the present invention should be distinguished fromconventional intra-articular injections of opiates and/or localanesthetics at the completion of arthroscopic or “open” joint (e.g.,knee, shoulder, etc.) procedures. The solution of the present inventionis used for continuous infusion throughout the surgical procedure toprovide preemptive inhibition of pain and inflammation. In contrast, thehigh concentrations necessary to achieve therapeutic efficacy with aconstant infusion of local anesthetics, such as lidocaine (0.5-2%solutions), would result in profound systemic toxicity.

Upon completion of the procedure of the present invention, it may bedesirable to inject or otherwise apply a higher concentration of thesame pain and inflammation inhibitors as used in the irrigation solutionat the operative site, as an alternative or supplement to opiates.

Some of the solutions of the present invention may suitably also includea gelling agent to produce a dilute gel. This gellable solution may beapplied, for example, to deliver a continuous, dilute localpredetermined concentration of agents.

VII. EXAMPLES

The following are several formulations in accordance with the presentinvention suitable for certain operative procedures followed by asummary of three clinical studies utilizing the agents of the presentinvention.

A. Example I

The following composition is suitable for use in anatomic jointirrigation during arthroscopic procedures. Each drug is solubilized in acarrier fluid containing physiologic electrolytes, such as normal salineor lactated Ringer's solution, as are the remaining solutions describedin subsequent examples.

Irrigation Solution for Arthroscopy

TABLE 24 Concentration Most (Nanomolar): Pre- Class of Agent DrugTherapeutic Preferred ferred serotonin₂ amitriptyline  100-50,0001,000-25,000  5,000 antagonist and histamine₁ antagonist cyclooxygenaseketoprofen 1,000-500,000 5,000-100,000 18,000 inhibitor mixed alpha-oxyme- 0.01-25,000 0.05-15,000 5,000 1/alpha-2 tazoline agonist(vasocon- strictor)

B. Example II Alternate Irrigation Solution for Arthroscopy

The following composition is also suitable for use in anatomic jointirrigation during arthroscopic procedures.

TABLE 25 Concentration Most (Nanomolar): Pre- Class of Agent DrugTherapeutic Preferred ferred mixed alpha- naphazoline  0.1-250,000  1-25,000 5,000 1/alpha-2 agonist (vasocon- strictor) serotonin₂amitriptyline  100-50,000 1,000-25,000  5,000 antagonist and histamine₁antagonist cyclooxygenase ketoprofen 1,000-500,000 5,000-100,000 20,000inhibitor

C. Example III Alternate Irrigation Solution for Arthroscopy

The following composition is also suitable for use in anatomicirrigation during arthroscopic procedures.

TABLE 26 Concentration (Nanomolar): Most Class of Agent Drug TherapeuticPreferred Preferred mixed alpha- oxyme- 0.01-25,000 0.05-15,000 5,0001/alpha-2 tazoline agonist (vasocon- strictor) Neurokinin₁ GR82334  1-1,000 10-500  100 antagonist cyclooxygenase ketoprofen 1,000-500,0005,000-100,000 20,000 inhibitor

D. Example IV Alternate Irrigation Solution for Arthroscopy

The following composition is also suitable for use in anatomicirrigation during arthroscopic procedures.

TABLE 27 Concentration (Nanomolar): Most Class of Agent Drug TherapeuticPreferred Preferred mixed alpha-1/ oxyme- 0.01-25,000 0.05-15,000 5,000alpha-2 agonist tazoline (vasoconstrictor) histamine₁ pyrilamine0.01-10,000 0.1-1,000   200 antagonist MAP kinase SB220025  0.05-100,000 0.2-25,000 20-5,000 inhibitor

E. Example V Alternate Irrigation Solution for Arthroscopy

The following composition is also suitable for use in anatomicirrigation during arthroscopic procedures.

TABLE 28 Concentration Most (Nanomolar): Pre- Class of Agent DrugTherapeutic Preferred ferred mixed alpha- oxyme- 0.01-25,000 0.05-15,0005,000 1/alpha-2 tazoline agonist (vasocon- strictor) serotonin₂amitriptyline  100-50,000 1,000-25,000  5,000 antagonist and histamine₁antagonist cyclooxygenase celecoxib 0.05-15,000 0.3-3,000 500 inhibitor

F. Example VI Inhibition of Synovial Plasma Extravasation by PreemptiveAdministration of an Anti-Inflammatory Irrigation Solution in the RatKnee

A rat knee joint model of acute inflammation (synovial plasmaextravasation) was studied to determine whether preemptiveintraarticular irrigation of the drugs ketoprofen, amitriptyline, oroxymetazoline, alone or in combination, can reduce inflammatorysolution-induced plasma extravasation. These three drugs, also used inthe exemplary solution of Example I above, were selected in part becauseof their abilities to collectively inhibit the inflammatory effects ofbiogenic amines, eicosanoid production, and the release of neuropeptidesfrom C-fiber terminals.

1. Methods

These studies were approved by the Committee on Animal Research of theUniversity of California, San Francisco. 104 male Sprague-Dawley rats(Bantin and Kingman, Fremont, Calif.) weighing 320 to 350 g were used.They were housed at 25° C. under controlled lighting conditions (lights6:00 AM to 6:00 PM) with food and water ad libitum.

Rats were anesthetized with sodium pentobarbital (65 mg/kgintraperitoneally; Abbott, Chicago, Calif.). The animals then received atail vein injection of Evans blue dye (50 mg/kg in a concentration of 20mg/mL; Sigma, St. Louis, Mo.), which was used as a marker for plasmaprotein extravasation. The knee joint capsule was exposed by excisingthe overlying skin, and a 30-gauge needle, which was used for theinfusion of fluid, was inserted into the joint. After perfusion of100-200 μL of fluid, a 25-gauge outflow needle was also placed into thejoint space approximately 3 mm from the inflow needle to extract fluid.The infusion and extraction rate (200 μL/min) was controlled by anSP120p push-pull syringe pump (WPI, Sarasota, Fla.). Perfusate sampleswere collected over 5-min intervals for 50 min. The samples wereimmediately centrifuged to determine whether red blood cells werepresent; only blood-free samples were acceptable. Samples were thenanalyzed for Evans blue dye concentration by spectrophotometricmeasurement of absorbance at 620 nm (Spectronic 21D; SpectronicInstruments, Inc., Rochester, N.Y.). Absorbance is linearly related todye concentration.

All drugs and chemicals, with the exception of mustard oil, were fromSigma, St. Louis, Mo. Plasma extravasation was activated by aninflammatory solution consisting of 1 μM 5-HT, 200 nM bradykinin, and 1%mustard oil (allyl isothiocyanate; Aldrich Chemical, Milwaukee, Wis.).Bradykinin and 5-HT were dissolved in saline, and mustard oil wasdissolved first in 40% ethanol and 20% Tween 80, with a finalconcentration of ethanol 1% and Tween 80 0.5%. Amitriptyline andoxymetazoline were dissolved in saline, and ketoprofen was dissolvedfirst in 40% ethanol and then diluted to a final ethanol concentrationof <1%.

To determine the optimal anti-inflammatory drug concentrations, each ofthe three drugs-amitriptyline, oxymetazoline, and ketoprofen-was testedfor its ability to dose-dependently inhibit plasma extravasationproduced by a single inflammatory mediator (5HT, mustard oil, andbradykinin, respectively) against which the drug is targeted. An initial5-min intraarticular baseline perfusion with 0.9% saline was followed bya 10-min perfusion with the drug, followed by a 35-min perfusion withthe drug and the inflammatory mediator.

In studies that tested the ability of the inflammatory solution tostimulate plasma extravasation, the inflammatory solution was perfusedfor 35 min, beginning immediately after a 15-min baseline salineperfusion. In experiments testing preemptive administration of thesingle drugs against the inflammatory solution, amitriptyline,oxymetazoline, or ketoprofen was added to the saline perfusion after 5min for a period of 10 min, then the drugs were perfused together withthe inflammatory solution for an additional 35 min. In addition toperfusing the individual drugs before, and then together with, theinflammatory solution, two-drug and three-drug combinations were alsotested for their ability to inhibit inflammatory solution-induced plasmaextravasation. Each of the four possible combinations(amitriptyline+ketoprofen, amitriptyline+oxymetazoline,ketoprofen+oxymetazoline, and amitriptyline+ketoprofen+oxymetazoline)were perfused 10 min before, and then together with, the inflammatorysolution, similar to the single-drug studies. In studies testing thepost-inflammatory administration of drugs, the inflammatory solutionalone was perfused for 10 min after the baseline 15-min salineperfusion, followed by all three anti-inflammatory drugs together withthe inflammatory solution for an additional 25 min.

A total of 68 rat knees were excluded from the study because of physicaldamage of the knee joint or inflow and outflow mismatch (detectable bypresence of blood in perfusate and high baseline plasma extravasationlevels or knee joint swelling caused by improper needle placement).After the procedure, rats were killed by a lethal injection ofpentobarbital and bilateral thoracotomy.

Data are expressed as the mean±SEM of absorbance at 620 nm for eachtreatment group. Data were analyzed using Total Absorbance (TotAbs),which was calculated by for each treatment group by taking the sum ofmean absorbance values over the time period from 5 minutes post-stimulus(T₀+5) to 35 minutes post-stimulus (T₀+35). Statistical analyses wereperformed using one-sided t-tests with a Bonferroni adjustment so thateach set of tests had level of significance 0.05.

2. Results

Amitriptyline was tested in concentrations of 20, 200, and 2000 nMagainst 5-HT-induced plasma extravasation (data not shown). An initial5-min baseline perfusion with 0.9% saline was followed by a 10-minperfusion with amitriptyline alone, followed by a 35min perfusion ofamitriptyline and 5-HT. Use of the absorbance measurement at each timepoint results in a calculated TotAbs. Amitriptyline 20 nM (n=6) has nosignificant effect on plasma extravasation levels produced by 5-HT 1 μM(n=9) (0.778±0.093 vs. 0.580±0.046; P>0.05). Amitriptyline 200 nM (n=6)inhibits 5-HT-induced plasma extravasation by 70% to 0.233±0.029(P<0.001). Amitriptyline 2000 nM (n=6) does not result in any furthersignificant decrease in plasma extravasation compared with the 200 nMconcentration (0.196±0.042; P>0.05, 2000 vs. 200 nM).

Similarly, oxymetazoline 20 nM (n=6) has no significant effect onmustard oil-induced plasma extravasation (1%; n=6) (1.555±0.266 vs.1.139±0.123; P>0.05) (data not shown). Oxymetazoline 200 nM (n=6)produces a 50% reduction of plasma extravasation (0.712±0.156; P=0.01).Oxymetazoline 2000 nM (n=6) does not result in any further significantdecrease in plasma extravasation compared with the 200 nM concentration(0.683±0.056; P>0.05, 2000 vs. 200 nM). The inhibitory activity ofketoprofen was tested against bradykinin (200 nM)-induced (n=6), plasmaextravasation at larger concentrations (75 to 7500 nM), given theextensive protein binding of ketoprofen in synovial fluid (25).Ketoprofen 75 nM (n=6) has no significant effect on bradykinin-inducedplasma extravasation (1.070±0.063 vs. 0.981±0.094; P>0.05), yetketoprofen 750 nM (n=6) reduces extravasation by 40% (0.673±0.079;P<0.005). Ketoprofen 7500 nM (n=6) does not produce any additionalinhibition compared with the 750 nM concentration (0.602±0.033; P>0.05,7500 nM vs. 750 nM) (data not shown). These dose-response data suggestthat amitriptyline 200 nM, oxymetazoline 200 nM, and ketoprofen 750 nMare concentrations that are maximally effective in inhibiting plasmaextravasation while avoiding unnecessarily large concentrations that mayproduce undesirable side effects. These concentrations were then used inthe following experiments.

Intraarticular perfusion of the inflammatory solution produces a markedincrease in plasma extravasation in the rat knee joint after 10 min andreaches a plateau by 15 min that continues for the remainder of theperfusion time (a total of 35 min) (FIG. 1). Use of the absorbancemeasurement at each time point results in a calculated TotAbs of4140±420 for the inflammatory solution alone. The perfusion of a singledrug-amitriptyline 200 nM (3430±140), oxymetazoline 200 nM (3330±170),or ketoprofen 750 nM (3080±260) 10 min before, and then in combinationwith, the inflammatory solution does not significantly inhibit plasmaextravasation.

The two-drug combinations also do not inhibit inflammatorysolution-induced plasma extravasation (FIG. 2). Amitriptyline+ketoprofen(2760±260), amitriptyline+oxymetazoline (2860±400), andketoprofen+oxymetazoline (2370±300) have TotAbs values not statisticallydifferent from inflammatory solution-induced plasma extravasation(4140±420). There are no significant differences among the two-drugcombinations.

The combination of all three drugs (amitriptyline, ketoprofen, andoxymetazoline) perfused 10 min before, and then in combination with, theinflammatory solution produces a dramatic inhibition of plasmaextravasation (1640±90; P<0.001) (FIG. 3). Compared with the two-drugcombinations, the three-drug combination is better at inhibiting plasmaextravasation than amitriptyline+ketoprofen (P<0.001),amitriptyline+oxymetazoline (P<0.01), or ketoprofen+oxymetazoline(P<0.05).

To determine the advantage of preemptively perfusing the three-drugsolution, the inflammatory solution was perfused for 10 min before theperfusion of the inflammatory solution+three-drug solution (FIG. 3). Theabove studies preperfused the drug solutions, whereas this study delaysthe addition of the drugs to the perfusion until 10 min after theadministration of the inflammatory solution has begun. The group withthe post-inflammatory three-drug administration shows significantly lessinhibition of plasma extravasation than that with the preemptiveadministration (P<0.01) and is not significantly different from theinflammatory solution alone. Therefore, the three-drug solution losesall ability to inhibit inflammatory solution-induced plasmaextravasation when perfused only 10 min later than the inflammatorysolution.

3. Discussion

Synovial perfusion of each one of the three drugs 10 min before, andthen in combination with, the inflammatory solution (bradykinin,5-hydroxytryptamine, and mustard oil) did not reduce plasmaextravasation. Similarly, two-drug combinations did not significantlyreduce inflammatory solution-induced plasma extravasation. Thecombination of all three drugs (amitriptyline, ketoprofen, andoxymetazoline) produced a dramatic inhibition of plasma extravasationand was more effective than any of the two-drug combinations. Acomparison between the preemptive (10 min before inflammatory solutionperfusion) and post-inflammatory administration (10 min afterinflammatory solution perfusion) showed that the post-inflammatoryadministration of the three-drug solution lost all ability to inhibitinflammatory solution-induced plasma extravasation.

This study demonstrated that a combination of the testedanti-inflammatory drugs including a vasoconstrictor delivered locallythroughout the duration of the inflammatory process was found to be theoptimal method for inhibiting the effects of peripherally actinginflammatory mediators.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes to the disclosedsolutions and methods can be made therein without departing from thespirit and scope of the invention. For example, alternate alpha-2adrenergic vasoconstrictors, pain inhibitors and anti-inflammationagents may be discovered that may augment or replace the disclosedagents in accordance with the disclosure contained herein. It istherefore intended that the scope of letters patent granted hereon belimited only by the definitions of the appended claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A composition for use inthe inhibition of pain at an anatomic site that can be adapted for localdelivery to the anatomic site during an operative, therapeutic ordiagnostic procedure, the composition comprising: oxymetazoline; acyclooxygenase inhibitor; and a pharmaceutical carrier, wherein thecomposition is suitable for adding to an irrigation solution for localdelivery at the anatomic site.
 2. The composition of claim 1, whereinthe cyclooxygenase inhibitor comprises ketoprofen.
 3. The composition ofclaim 1, further comprising amitriptyline.
 4. The composition of claim1, wherein the pharmaceutical carrier comprises a liquid irrigationcarrier.
 5. A solution formulated for use in the inhibition of pain atan anatomic site that can be adapted for local delivery to the anatomicsite during an operative, therapeutic or diagnostic procedure, thesolution comprising: oxymetazoline and amitriptyline in a pharmaceuticalcarrier, wherein the composition is suitable for adding to an irrigationsolution for local delivery at the anatomic site.
 6. The solution ofclaim 5, wherein the solution further comprises an additionalpain/inflammation inhibitory agent.
 7. A solution formulated for use inthe inhibition of pain at an anatomic site that can be adapted for localdelivery to the anatomic site during an operative, therapeutic ordiagnostic procedure, the solution comprising: at least onevasoconstrictive agent that is an a-adrenergic receptor agonist selectedfrom the group consisting of oxymetazoline, p-aminoclonidine,naphazoline, tetrahydrozoline, anatazoline, tramazoline, monoxidine,apraclonidine, guanfacine, guanabenz and xylazine; at least a firstpain/inflammation inhibitory agent selected to act on a differentmolecular target than the a-adrenergic receptor agonist; and a liquidcarrier, wherein the solution is suitable for adding to an irrigationsolution for local delivery at the anatomic site during the procedure.8. A composition for use in the inhibition of pain at an anatomic sitethat can be adapted for local delivery to the anatomic site during anoperative, therapeutic or diagnostic procedure, the compositioncomprising: oxymetazoline; at least a first pain/inflammation inhibitoryagent selected to act on a different molecular target than aα-adrenergic receptor, wherein the first pain/inflammation inhibitoryagent is selected from the group consisting of cyclooxygenaseinhibitors, serotonin receptor antagonists and histamine receptorantagonists; and a pharmaceutical carrier, wherein the composition issuitable for adding to an irrigation solution for local delivery at theanatomic site.
 9. A solution formulated for use in the inhibition ofpain at a joint of an extremity that can be adapted for local deliveryto the joint during an operative, therapeutic or diagnostic procedure,the solution comprising: oxymetazoline, amitriptyline and ketoprofen ina liquid carrier.